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This study was aimed to evaluate the influence of Human Leukocyte Antigens- G (HLA-G) gene polymorphisms on the risk of rheumatoid arthritis (RA). In the present study, blood samples were collected from 100 rheumatoid arthritis patients and 100 healthy controls. Serum levels of HLA-G in both patients and controls were measured using ELISA kit which was shown a higher significant differences (p< 0.05) in comparison between them (539.3 ng/ml and 139.3ng/ml) respectively. The frequency of the allele and genotypes in the patient groups and control groups were determined using Polymerase Chain Reaction (PCR) for 14 bp polymorhisms in exon 8 (rs66554220) while direct sequencing analysis was done to determine exon 2 (rs1130355) (G 1850 A) SNPs of human leucocyte antigens- G (HLA- G). There was statistically significant difference in all genotypes of HLA-G14-bp of exon 8 in RA patients in comparison with controls. The probability of homozygote genotype of insertion was (0.006), homozygote genotype of deletion was (0.003) and for heterozygote of insertion was (0.000) in comparison between RA patients and controls. From the results of odd ratio (OR) found that existence of homozygotes of insertion give protection effect from disease O.R= 0.44 while heterozygotes of deletion act as risk factor for disease O.R= 3.31. Homozygotes of deletion had no effect because was not found in patients groups. From the sequencing results of exon 2 of HLA-G gene found the mutation occur in position 1850 G>A which give three genotypes (GG, AG and AA). Calculations of genotypes frequency for exon 2 showed that GG and AA genotypes was associated with the disease with p- value (0.000) for both genotypes. While AG genotype showed no association with disease p value (0.449).From the results of odd ratio found that GG and AA of exon 2 gave protection effect from disease O.R( 0.29 and 0.76 ) respectively, while AG genotype acted as risk factor for disease O.R= 4.0. [ABSTRACT FROM AUTHOR]

Pathogenic variants in the Parkin-gene (PRKN) are among the most common genetic causes of early onset Parkinson's disease (EOPD). Patients with EOPD can present with atypical clinical features and misdiagnosis is frequent. Here, we report a clinical phenotype with atypical signs and symptoms of a 35-year-old male patient with EOPD caused by a compound heterozygous PRKN-gene deletion of exons 2 and 4. After the initial diagnosis of stiff person syndrome, the patient was admitted to our department for a second opinion after 8 years of untreated disease progression. The patient presented with prominent spastic paraparesis pronounced on the right side and hyperreflexia as well as Parkinsonism with rigidity predominantly affecting the upper limbs, bradykinesia, and resting tremor. In the diagnostic assessment, magnetic evoked potentials to the anterior tibial muscles showed a low amplitude on the right side, compatible with pyramidal tract disturbance. However, an MRI of the head and the spine did not show any pathologies or atrophy. A [123I] FP-CIT SPECT scan revealed profoundly and left-pronounced reduced striatal uptake suggesting a neurodegenerative Parkinson's syndrome. Even though an acute levodopa challenge did not show marked improvement of symptoms, the chronic levodopa challenge with up to 450 mg/day significantly reduced the rigidity and bradykinesia. Surprisingly, spastic paraparesis and hyperreflexia diminished under dopaminergic treatment. Finally, genetic analysis by next-generation sequencing via copy number variant analysis (CNV) and multiplex ligation-dependent probe amplification (MLPA) confirmed compound heterozygous deletions of exons 2 and 4 in the PRKN-gene. As presented in this case, the awareness of atypical clinical symptoms of EOPD is essential to prevent misdiagnosis in young patients.

Background: Leptin hormone, encoded by leptin (LEP) gene is involved in many biological and physiological processes in the body. Polymorphism in LEP gene has been observed and correlated with a variety of reproductive and productive traits in several sheep breeds worldwide, but its role has not been much studied in local sheep breeds of Pakistan. The present study was conducted to analyze polymorphism in LEP gene in Lohi breed of sheep. Methods: Subsequent to statistical analysis (generalized linear model), 18 animals were selected randomly from the flock for blood samples collection followed by DNA extraction, amplification using PCR prior to sequencing. The amplified product of exon 2 and partial intron 2 regions of LEP gene was 268bp. Results: Molecular analysis showed a heterozygous condition i.e. C>Y at position 15 and 18 in exon 2. The data on average daily weight gain (ADG) from birthday to 90 days were used for association study, while environmental effects were minimized by means of generalized linear model. Association of polymorphisms in LEP gene with ADG did not yield any significant results. Conclusion: In conclusion, analysis of LEP gene sequence verified the existence of genetic changes in Lohi sheep. Further investigations are needed to find variations that might be linked with traits of economic importance for upcoming breeding program sand marker-assisted selection.

Abstract Background Tumor suppressor gene p16 promoter hypermethylation has been widely studied in colorectal cancer (CRC), yet its clinicopathological significance remains controversial. The methylation alterations of other regions within p16 gene are still rarely researched. The present study aimed to explore the methylation changes of p16 gene body in CRC and to find whether they were associated with clinicopathological staging of CRC. Methods Paired colorectal cancer tissues and corresponding adjacent normal tissues from 30 CRC patients were collected. The methylation levels of two CpG islands within p16 gene body, exon 1 and exon 2, were accurately assessed simultaneously by a LC-MS/MS method. The p16 protein expressions were assessed by immunohistochemistry assay. Statistical analyses were carried out using SPSS 17.0 software. Heat-map analysis was carried out by HemI 1.0 software. Results In the present study, CRC tissues showed more highly methylated than adjacent normal tissues at both CpG islands of p16 gene. And exon 2 hypermethylation was higher and more frequent than exon 1. The ROC curve analysis showed that the simultaneous use of both indicators had excellent sensitivity and specificity for distinguishing CRC tissues and adjacent normal tissues. Following, the methylation level of p16 exon 1/2 was negatively related to p16 protein expression. Further correlation analysis revealed that p16 exon 1 hypermethylation was associated with N/Dukes staging (p = 0.033), and p16 exon 2 hypermethylaiton was associated with T staging (p = 0.035). Conclusions The p16 gene body was remarkably hyper-methylated in CRC tissues and associated with p16 protein expression and cancer clinicopathological staging. The combination of p16 exon 1 and exon 2 could better reflect the overall methylation status of p16 gene body and provide potential biomarkers of CRC.

Abstract Background Breast carcinogenesis is a multistep process involving genetic and epigenetic changes. Tumor tissues are frequently characterized by gene-specific hypermethylation and global DNA hypomethylation. Aberrant DNA methylation levels have, however, not only been found in tumors, but also in tumor-surrounding tissue appearing histologically normal. This phenomenon is called field cancerization. Knowledge of the existence of a cancer field and its spread are of clinical relevance. If the tissue showing pre-neoplastic lesions is not removed by surgery, it may develop into invasive carcinoma. Methods We investigated the prevalence of gene-specific and global DNA methylation changes in tumor-adjacent and tumor-distant tissues in comparison to tumor tissues from the same breast cancer patients (n = 18) and normal breast tissues from healthy women (n = 4). Methylation-sensitive high resolution melting (MS-HRM) analysis was applied to determine methylation levels in the promoters of APC, BRCA1, CDKN2A (p16), ESR1, HER2/neu and PTEN, in CDKN2A exon 2 and in LINE-1, as indicator for the global DNA methylation extent. The methylation status of the ESR2 promoter was determined by pyrosequencing. Results Tumor-adjacent and tumor-distant tissues frequently showed pre-neoplastic gene-specific and global DNA methylation changes. The APC promoter (p = 0.003) and exon 2 of CDKN2A (p

Objective: The P21waf is a tumor-suppressor protein encoded by CDKN1A gene. In this study, we evaluated P21waf expression and CDKN1A exon 2 mutation and their relationships with clinicopathological parameters and cancer development in salivary adenoid cystic carcinoma (ACC). Patients and Methods: Forty paraffin blocks from patients with salivary ACC were collected. Immunohistochemical staining was performed using P21 antibody. Genomic DNAs were extracted from the deparaffinized sections of the embedded tissue. Exon 2 of CDKN1A gene was amplified by PCR and the PCR products were sequenced. Spearman’s correlation coefficient, Fisher’s exact test, and Kruskal-Wallis test were used for data analysis. Results: A significant inverse correlation was observed between P21 expression and histologic grade (p=0.033, r=-0.338). The correlation of tumor size with recurrence (p=0.048) and tumor stage (p=0.046) was also evidenced. No mutation was detected in the exon 2 of CDKN1A gene. Conclusions: Regarding the association of P21 expression and histologic grade as a major prognostic indicator of ACC, P21 may be a useful prognostic indicator in ACC. On the other hand, CDKN1A exon 2 mutation seems inapplicable as a risk factor for ACC development.

Exon 2 of MHC class II gene codes for the first domain of the molecule that forms the peptide‐binding groove and its polymorphism partly explains functional MHC diversity. A 850 bp DQA1 gene fragment spanning from intron I to exon III was typed by sequencing of 40 Tharparkar cattle of various agro‐climatic zones of northern India along with 10 Tharparkar crossbreds. On analysis of nucleotide sequences, a total of 30 polymorphic sites (1 insertion and 29 SNPs) were identified in 14 MHC alleles leading to amino acid changes in 5 places in 249 bp (exon 2). Five new BoLa DQA1 alleles were identified and reported. The within group mean distance was highest in Tharparkar herd of Bikaner (0.045) and lowest (0.020) in that of Surathgarh (breeding tract) whereas, between groups mean distance was highest in Bikaner Tharparkar—Suratgarh Tharparkar pair. There was excess of nonsynonymous over synonymous nucleotide substitutions in the present study. The effects of these substitutions were predicted using I‐Mutant and Panther online resources. The mean ratio of dN/dS was found to be >1.0 at 12 codons with two mutation hotspots at 13th codon (P = 0.002) and 64th codon (P = 0.01). The phylo‐geographic analysis revealed that alleles 5, 7 and 13 formed a different cluster with alleles 7 and 13 grouped by the most frequent allele (BoLa‐DQA*1401). [ABSTRACT FROM AUTHOR]

Studies performed on farm animals like cattle and pig have shown that there has been a relationship between leptin gene (LEP) and carcass meat quality, milk production and content, and economic parameters such as reproduction and food consumption. There has been scarce research conducted related to leptin gene of sheep and especially goat. The aim of the study is to reveal the genetic structures of goats living in Turkey through nucleotide sequence analysis in targeted zones of LEP gene Exon 2 and Intron 2 of Anatolian Black, Kilis and Angora goat breeds which are commonly fed in Anatolia. According to the sequence analysis results of each three breeds, Anatolian Black goat breed have the highest haplotype number with nucleotide and haplotype diversity both in exon 2 and intron 2. There was only one haplotype found in both exon 2 and intron 2 in Angora goat breed. There was no nucleotide diversity found in individuals belonging to Angora goat breed. Taking the regions analyzed for LEP gene into consideration, it is seen that Anatolian Black goat breed has the highest genetic diversity among other goat breeds fed in Anatolia. Future studies upon the LEP gene in goats should take into account of increasing the sample size and of base in order to obtain more useful information for better understanding the gene structure.

The tyrosine kinase receptor RET, which is the protein product of the RET gene, is involved in the development of the mammalian nervous system that causes Hirschsprung’s disease (HSCR). RETs are cell surface molecules that are expressed in cells derived from the neural crest. The purpose of this study was to investigate the polymorphism of the RET gene in HSCR in the Yogyakarta population. Genomic DNA was extracted from surgically removed bowel tissues of 54 unrelated HSCR patients. Exon 2 of the RET gene was amplified by polymerase chain reaction (PCR) and analyzed by restriction fragment length polymorphism (RFLP). Molecular results were compared with clinical performance of Hirschsprung patients. RET polymorphism was detected in exon 2 in all of the 54 Indonesian HSCR patients. The allelic distribution of the c135GàA polymorphism in the RET exon 2 indicated that the A allele was more frequent in patients than in control individuals (chi-square test, p= 0.001). Thus the RET variant allele A is over-represented in patients affected with the HSCR phenotype. Polymorphism of exon 2 of the RET gene was found in sporadic Hirschsprung’s disease in the Yogyakarta population, which suggests that the RET gene plays important roles in the pathogenesis of HSCR.

Objective: Familial Mediterranean fever (FMF) is the most common autoinflammatory disease. Most of the identified disease-causing mutations are located on exon 10. As the number of studies about the effect of the exonal location of the mutation and its phenotypic expression is limited, we aimed to investigate whether the exonic location of the Mediterranean fever (MEFV) mutation has an effect on the clinical manifestation in patients with FMF. Methods: Study population was derived from the main FMF registry that included 2246 patients from 15 different rheumatology clinics. We categorized the mutations according to their exon locations and retrieved the clinical and demographic information from the database. Results: Patients having the MEFV mutations on exon 2 or 10 (n:1526) were divided into three subgroups according to the location of the MEFV mutations: Group 1 (exon 2 mutations), Group 2 (exon 10 mutations), and Group 3 (both exon 2 and exon 10 mutations). Group 2 patients were of a significantly younger age at onset, and erysipel-like erythema, arthritis, amyloidosis, and a family history of FMF were more common in this group. Conclusion: Patients with FMF and exon 10 mutations show more severe clinical symptoms and outcome. Exon 2 mutations tend to have a better outcome. [ABSTRACT FROM AUTHOR]

Objective: Familial Mediterranean fever (FMF) is the most common autoinflammatory disease. Most of the identified disease-causing mutations are located on exon 10. As the number of studies about the effect of the exonal location of the mutation and its phenotypic expression is limited, we aimed to investigate whether the exonic location of the Mediterranean fever (MEFV) mutation has an effect on the clinical manifestation in patients with FMF. Methods: Study population was derived from the main FMF registry that included 2246 patients from 15 different rheumatology clinics. We categorized the mutations according to their exon locations and retrieved the clinical and demographic information from the database. Results: Patients having the MEFV mutations on exon 2 or 10 (n:1526) were divided into three subgroups according to the location of the MEFV mutations: Group 1 (exon 2 mutations), Group 2 (exon 10 mutations), and Group 3 (both exon 2 and exon 10 mutations). Group 2 patients were of a significantly younger age at onset, and erysipel-like erythema, arthritis, amyloidosis, and a family history of FMF were more common in this group. Conclusion: Patients with FMF and exon 10 mutations show more severe clinical symptoms and outcome. Exon 2 mutations tend to have a better outcome. [ABSTRACT FROM AUTHOR]

Abstract The amino acid substitution at residue 76 of the food vacuolar transmembrane protein encoded by the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) is an important, albeit imperfect, determinant of chloroquine susceptibility status of the parasite. Other mutations in Pfcrt can modulate susceptibility of P. falciparum to other antimalarials capable of interfering with heme detoxification process, and may exert compensatory effect on parasite growth rate. To address whether nationwide implementation of artemisinin combination therapy (ACT) in Thailand could affect sequence variation in exon 2 and introns of Pfcrt , we analyzed 136 P. falciparum isolates collected during 1997 and 2016 from endemic areas bordering Myanmar, Cambodia and Malaysia. Results revealed 6 haplotypes in exon 2 of Pfcrt with 2 novel substitutions at c.243A > G (p.R81) and c.251A > T (p.N84I). Positive selection was observed at amino acid residues 75, 76 and 97. Four, 3, and 2 alleles of microsatellite (AT/TA) repeats occurred in introns 1, 2 and 4, respectively, resulting in 7 different 3-locus haplotypes. The number of haplotypes and haplotype diversity of exon 2, and introns 1, 2 and 4 were significantly greater among isolates collected during 2009 and 2016 than those collected during 1997 and 2008 when 3-day ACT and 2-day ACT regimens were implemented nationwide, respectively (p < 0.05). By contrast, the number of haplotypes and haplotype diversity of the merozoite surface proteins 1 and 2 of these parasite populations did not differ significantly between these periods. Therefore, the Pfcrt locus of P. falciparum in Thailand continues to evolve and could have been affected by selective pressure from modification of ACT regimen. Highlights • Sequence variation in exon 2 and introns of Pfcrt occurred among P. falciparum in Thailand. • The number of haplotypes and haplotype diversity of Pfcrt increased during nationwide 3-day ACT. • Modification of ACT duration may affect evolution of Pfcrt in Thailand. [ABSTRACT FROM AUTHOR]

Background: Tumor suppressor gene p16 promoter hypermethylation has been widely studied in colorectal cancer (CRC), yet its clinicopathological significance remains controversial. The methylation alterations of other regions within p16 gene are still rarely researched. The present study aimed to explore the methylation changes of p16 gene body in CRC and to find whether they were associated with clinicopathological staging of CRC.Methods: Paired colorectal cancer tissues and corresponding adjacent normal tissues from 30 CRC patients were collected. The methylation levels of two CpG islands within p16 gene body, exon 1 and exon 2, were accurately assessed simultaneously by a LC-MS/MS method. The p16 protein expressions were assessed by immunohistochemistry assay. Statistical analyses were carried out using SPSS 17.0 software. Heat-map analysis was carried out by HemI 1.0 software.Results: In the present study, CRC tissues showed more highly methylated than adjacent normal tissues at both CpG islands of p16 gene. And exon 2 hypermethylation was higher and more frequent than exon 1. The ROC curve analysis showed that the simultaneous use of both indicators had excellent sensitivity and specificity for distinguishing CRC tissues and adjacent normal tissues. Following, the methylation level of p16 exon 1/2 was negatively related to p16 protein expression. Further correlation analysis revealed that p16 exon 1 hypermethylation was associated with N/Dukes staging (p = 0.033), and p16 exon 2 hypermethylaiton was associated with T staging (p = 0.035).Conclusions: The p16 gene body was remarkably hyper-methylated in CRC tissues and associated with p16 protein expression and cancer clinicopathological staging. The combination of p16 exon 1 and exon 2 could better reflect the overall methylation status of p16 gene body and provide potential biomarkers of CRC. [ABSTRACT FROM AUTHOR]

Background: Breast carcinogenesis is a multistep process involving genetic and epigenetic changes. Tumor tissues are frequently characterized by gene-specific hypermethylation and global DNA hypomethylation. Aberrant DNA methylation levels have, however, not only been found in tumors, but also in tumor-surrounding tissue appearing histologically normal. This phenomenon is called field cancerization. Knowledge of the existence of a cancer field and its spread are of clinical relevance. If the tissue showing pre-neoplastic lesions is not removed by surgery, it may develop into invasive carcinoma.Methods: We investigated the prevalence of gene-specific and global DNA methylation changes in tumor-adjacent and tumor-distant tissues in comparison to tumor tissues from the same breast cancer patients (n = 18) and normal breast tissues from healthy women (n = 4). Methylation-sensitive high resolution melting (MS-HRM) analysis was applied to determine methylation levels in the promoters of APC, BRCA1, CDKN2A (p16), ESR1, HER2/neu and PTEN, in CDKN2A exon 2 and in LINE-1, as indicator for the global DNA methylation extent. The methylation status of the ESR2 promoter was determined by pyrosequencing.Results: Tumor-adjacent and tumor-distant tissues frequently showed pre-neoplastic gene-specific and global DNA methylation changes. The APC promoter (p = 0.003) and exon 2 of CDKN2A (p < 0.001) were significantly higher methylated in tumors than in normal breast tissues from healthy women. For both regions, significant differences were also found between tumor and tumor-adjacent tissues (p = 0.001 and p < 0.001, respectively) and tumor and tumor-distant tissues (p = 0.001 and p < 0.001, respectively) from breast cancer patients. In addition, tumor-adjacent (p = 0.002) and tumor-distant tissues (p = 0.005) showed significantly higher methylation levels of CDKN2A exon 2 than normal breast tissues serving as control. Significant correlations were found between the proliferative activity and the methylation status of CDKN2A exon 2 in tumor (r = -0.485, p = 0.041) and tumor-distant tissues (r = -0.498, p = 0.036).Conclusions: From our results we can conclude that methylation changes in CDKN2A exon 2 are associated with breast carcinogenesis. Further investigations are, however, necessary to confirm that hypermethylation of CDKN2A exon 2 is associated with tumor proliferative activity. [ABSTRACT FROM AUTHOR]

The tyrosine kinase receptor RET, which is the protein product of the RET gene, is involved in the development of the mammalian nervous system that causes Hirschsprung’s disease (HSCR). RETs are cell surface molecules that are expressed in cells derived from the neural crest. The purpose of this study was to investigate the polymorphism of the RET gene in HSCR in the Yogyakarta population. Genomic DNA was extracted from surgically removed bowel tissues of 54 unrelated HSCR patients. Exon 2 of the RET gene was amplified by polymerase chain reaction (PCR) and analyzed by restriction fragment length polymorphism (RFLP). Molecular results were compared with clinical performance of Hirschsprung patients. RET polymorphism was detected in exon 2 in all of the 54 Indonesian HSCR patients. The allelic distribution of the c135GàA polymorphism in the RET exon 2 indicated that the A allele was more frequent in patients than in control individuals (chi-square test, p= 0.001). Thus the RET variant allele A is over-represented in patients affected with the HSCR phenotype. Polymorphism of exon 2 of the RET gene was found in sporadic Hirschsprung’s disease in the Yogyakarta population, which suggests that the RET gene plays important roles in the pathogenesis of HSCR.

OBJECTIVE: The present study evaluated the association between lactotransferrin (LTF) gene polymorphism (exon 2, A/G, Lys/Arg) and dental caries. MATERIAL AND METHODS: A convenience sample of 110 individuals, 12 years old, was divided into: group 1, 48 individuals without caries experience (DMFT=0), and group 2, 62 subjects with caries experience (DMFT>1). DNA was obtained from a mouthwash with 3% glucose solution, followed by a scrapping of the oral mucosa. After DNA purification, polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) was performed to access the study polymorphism. The LTF A/G (Lys/Arg) polymorphism had been previously reported as located in exon 1. RESULTS: Allele 1 of the study polymorphism was associated with low DMFT index and showed a protective effect against caries experience (OR=0.16, IC=0.03-0.76, p=0.01). CONCLUSIONS: Lactotransferrin A/G (exon 2, Lys/Arg) polymorphism was associated with susceptibility to dental caries in 12-year-old students.

Background: The Major Histocompatibility Complex (MHC) is a genomic region containing genes with crucial roles in immune responses. MHC class I and class II genes encode antigen-presenting molecules expressed on the cell surface. To counteract the high variability of pathogens, the MHC evolved into a region of considerable heterogeneity in its organization, number and extent of polymorphism. Studies of MHCs in different model species contribute to our understanding of mechanisms of immunity, diseases and their evolution. Camels are economically important domestic animals and interesting biomodels. Three species of Old World camels have been recognized: the dromedary (Camelus dromedarius), Bactrian camel (Camelus bactrianus) and the wild camel (Camelus ferus). Despite their importance, little is known about the MHC genomic region, its organization and diversity in camels. The objectives of this study were to identify, map and characterize the MHC region of Old World camelids, with special attention to genetic variation at selected class MHC II loci. Results: Physical mapping located the MHC region to the chromosome 20 in Camelus dromedarius. Cytogenetic and comparative analyses of whole genome sequences showed that the order of the three major sub-regions is "Centromere - Class II - Class III - Class I". DRA, DRB, DQA and DQB exon 2 sequences encoding the antigen binding site of the corresponding class II antigen presenting molecules showed high degree of sequence similarity and extensive allele sharing across the three species. Unexpectedly low extent of polymorphism with low numbers of alleles and haplotypes was observed in all species, despite different geographic origins of the camels analyzed. The DRA locus was found to be polymorphic, with three alleles shared by all three species. DRA and DQA sequences retrieved from ancient DNA samples of Camelus dromedarius suggested that additional polymorphism might exist. Conclusions: This study provided evidence that camels possess an MHC comparable to other mammalian species in terms of its genomic localization, organization and sequence similarity. We described ancient variation at the DRA locus, monomorphic in most species. The extent of molecular diversity of MHC class II genes seems to be substantially lower in Old World camels than in other mammalian species. [ABSTRACT FROM AUTHOR]

Hearing loss (HL) is one of the most common human defects which affects millions of people globally. The identification of deafness-related genes or loci may facilitate basic and clinical translational research on this disorder. Here, we investigated a Chinese family with autosomal recessive non-syndromic hearing impairment. Using targeted massively parallel sequencing, we identified a novel homozygous mutation, c.3525_3526insA and p.Q1175fsX1188 (NM_016239), in exon 2 of MYO15A . Sanger sequencing confirmed that affected siblings were homozygous for the mutation, whereas both normal hearing parents were heterozygous. The mutation was absent in 96 healthy controls and public databases. The insertion leads to a frameshift and a truncated form of the protein, resulting in the pathogenic effect of hearing loss for the patients. Mutations in exon 2 of MYO15A may cause a less severe phenotype, facilitating the rapid identification of mutations in exon 2 among the 66 exons when linkage of less severe hearing loss to Deafness, Autosomal Recessive 3 (DFNB3) is detected. Our data provide additional molecular information for establishing a better genotype–phenotype understanding of DFNB3. [ABSTRACT FROM AUTHOR]

In the treatment of metastatic colorectal cancer, chemotherapy is used as a backbone to which an angiogenesis inhibitor such as bevacizumab (vascular endothelial growth factor [VEGF]) or a monoclonal antibody to the epidermal growth factor receptor (EGFR) such as cetuximab or panitumumab is added. In this article, we seek answers to the question: 'FIRE-3 and C80405: is there a best choice for initial treatment of metastatic colorectal cancer?' In the absence of clear and univocal evidence that EGFR inhibitors should be used in the first-line setting for patients with metastatic RAS wild-type colorectal cancer, the physician and patient still have the choice between bevacizumab or an EGFR inhibitor as addition to the chemotherapy (FOLFIRI or FOLFOX) in this setting. This choice will be influenced by the different toxicity profiles of bevacizumab and cetuximab or panitumumab and will therefore depend on existing comorbidities of the patient and patient/physician preferences. The higher response rate of the combination of chemotherapy with cetuximab could make this treatment more interesting as induction therapy. [ABSTRACT FROM AUTHOR]

Background Familial Mediterranean Fever (FMF), characterized by recurrent fever and inflammation of serous membranes, is an autosomal recessive disease caused by mutations in the Mediterranean fever (MEFV) gene. Around 296 mutations have been reported to date. Methods Two two-generation Turkish families with a total of four members diagnosed with FMF clinically were screened with DNA sequencing performed on exon 2 and exon 10 of the MEFV genes. Then, complete exome sequencing analysis of MEFV gene was done for four patients in whom novel mutation was detected. Results A novel single base Guanine (G) insertion mutation in the coding region of MEFV gene, named c.2330dupG (p.Gln778Serfs*4 or Q778SfsX4) resulting in a mutated Pyrin/Marenostrin protein was identified. Conclusions This is the first report of a new mutation in exon 10 of the MEFV gene in two Turkish families. This novel pattern of insertion mutation may provide important information for further studies on FMF pathogenesis. [ABSTRACT FROM AUTHOR]

Over the years, deletions of CDKN2A (p16) tumor suppressor gene has been studied using FISH and multiplex PCR, with major focus on exon 2 in various cancers, and the frequency of mutation is found to be varied in different studies. In this study, we analyzed the deletion status of all three exons of p16 and frequency of exon 2 somatic point mutations in glioma from the Indian population and its clinical implications. Multiplex PCR was carried out in order to check deletion of all 3 exons in 50 glioma samples. Nonconventional PCR-SSCP analysis and sequencing was done to identify mutations in 48 cases. Deletion of at least one of the three exons of p16 INK4A was observed in ten cases (20 %). The frequencies of exon-wise deletions were 10 % for exon 1, 4 % for exon 2, and 8 % for exon 3. Two out of 48 samples were positive for mutations in p16 exon 2. One sample had a transition of G to C on position 147 with a codon change TGG to TGC which does not contribute to the protein structure. Another sample had a transversion of A to G on the position 154 with a codon change ATG to GTG with change in amino acid methionine to valine in 52nd position. Deletion pattern was found to be varied in three exons. Frequency of p16 gene mutation was less in the Indian population (4.2 %), and this mutation does not contribute to any remarkable change in protein structure. [ABSTRACT FROM AUTHOR]

The DQB1 locus is located in the major histocompatibility complex (MHC) class II region and involved in immune response. We identified 20 polymorphic sites in a 228 bp fragment of exon 2, one of the most critical regions of the MHC DQB1 gene, in 60 Nigerian goats. Four sites are located in the peptide binding region, and 10 amino acid substitutions are peculiar to Nigerian goats, compared with published sequences. A significantly higher ratio of nonsynonymous/synonymous substitutions ( dN/ dS) suggests that allelic sequence evolution is driven by balancing selection ( P < 0.01). In silico functional analysis using PANTHER predicted that substitution P56R, with a subPSEC score of −4.00629 (P deleterious = 0.73229), is harmful to protein function. The phylogenetic tree from consensus sequences placed the two northern breeds closer to each other than either was to the southern goats. This first report of sequence diversity at the DQB1 locus for any African goat breed may be useful in the search for disease-resistant genotypes. [ABSTRACT FROM AUTHOR]

ABSTRACT Lipoid proteinosis (LP) is a rare disorder characterized by extensive hyaline-like deposits on the skin, mucous membranes and various internal organs with varying clinical manifestations. The disorder has been recently shown to result from loss-of-function mutations in the extracellular matrix protein 1 gene (ECM1) on 1Q21. The two cases reported here had typical clinical and histological features consistent with LP. Direct sequencing of amplified DNA from the second patient showed a single nucleotide substitution (C > T) at nucleotide 94 within exon 2 of the ECM1 gene, nonsense mutation Q32X. This is the second case reported of LP with involvement of exon 2 of ECM1. [ABSTRACT FROM AUTHOR]

Abstract: Abnormal tau cleavage is prominent in the neurofibrillary degeneration characteristic of Alzheimer’s disease (AD) and related tauopathies. We recently showed that cleaved human tau is secreted by specific mechanisms when overexpressed. Here we examined the effect of expressing N-terminal and full length tau constructs in transiently and stably transfected neuronal lines. We show that secreted tau exhibits a cleavage pattern similar to CSF-tau from human AD patients and that tau secretion is specifically inhibited by the presence of the exon 2 insert. These results suggest that tau secretion may play a hitherto unsuspected role in AD and related tauopathies. [Copyright &y& Elsevier]

An experiment was conducted using randomly selected 150 hens ofiWN strain of White Leghorn maintained at AICRP on Poultry Improvement, Mannuthy to identify the SNP in the exon 2 of ovocalyxin-32 gene and its association with the production traits. PCR-RFLP analysis was done to find the genotypes TT, TG and GG in exon 2. The genotypic frequencies of TT, TG and GG were 0.900, 0.047 and 0.053, respectively and the allelic frequencies of T and G were 0.92 and 0.08, respectively. The frequency of normal allele Twas much higher than the mutant G. The result of this study revealed no significant association of c.267T>G with body weight, egg weight, egg number, shell thickness and age at sexual maturity. Present study indicates that the SNP in the exon 2 of ovocalyxin-32 (OCX-32) can't be used as a marker for selection of IWN strain of White Leghorn for eggs. [ABSTRACT FROM AUTHOR]

Major histocompatibility complex (MHC) II genes are key factors in presenting antigens derived from extracellular pathogens to CD4+ T cells. These genes are known to be associated with disease resistance in livestock, including cultured fish species. Genome-wide linkage analysis of RSIV-resistant red sea bream has revealed that MHC IIβ is linked to the RSIV-resistant trait and was suggested as a probable candidate gene responsible for the resistant trait. In the present study, diversity of the MHC IIβ gene in wild and farmed red sea bream populations was evaluated by analysis of an intronic minisatellite locus, PmaMHC2b. Haplotypes of the βI domain of MHC IIβ were identified by cloning, and linkage of minisatellite polymorphisms with haplotype was confirmed in farmed populations. Large numbers of alleles were observed in the wild population (30), while the domesticated populations showed significantly lower values (13). Thirteen haplotypes of MHC IIβ were observed; most haplotypes possessed the unique PmaMHC2b allele, but some were present in more than one PmaMHC2b allele. Each haplotype possessed a distinct amino acid sequence with no synonymous mutations. Among 16 amino acid positions involved in peptide-binding grooves, four were monomorphic and 12 polymorphic. Several positions differed in the RSIV-resistant and RSIV-susceptible phenotypes: five in hydrophilic/hydrophobic character and five in polarity. These results provide insight applicable to broodstock management and contribute to understanding RSIV resistance. • Polymorphism of MHC IIβ gene was analyzed in wild and cultured red sea bream. • Diversity of MHC IIβ gene was significantly lower in farmed populations. • Thirteen haplotypes of the β1 domain of MHC IIβ were identified in farmed populations. • One haplotype was significantly linked to RSIV resistance. [ABSTRACT FROM AUTHOR]

We provide the first description and confirmation of expression for three Major Histocompatibility Complex (MHC) genes (DQA, DQB, and class I) in the New Zealand endemic Hector's dolphin ( Cephalorhynchus hectori). Sequences were derived from cDNA generated from peripheral blood leukocyte mRNA collected during a temporary live capture of two Hector's dolphins. The complete exons 2 and 3 of DQA, DQB, and class I were amplified from cDNA to confirm their expression. The same primers proved reliable for amplification from genomic DNA. Locus identity was supported by phylogenetic relationships to MHC molecules from other closely related species. No evidence was found for duplication of DQ loci, but cloning of amplified exon 2 of the class I gene indicated duplication or triplication of this locus. However, the expression of multiple class I loci was uncertain as only one allele could be detected from clones of cDNA. Balancing selection was indicated by a significantly greater ratio of nonsynonymous to synonymous substitutions at all three loci and by the apparent transspecific sharing of alleles for DQA and DQB. [ABSTRACT FROM AUTHOR]

The variation of the exon 2 of the major histo-compatibility complex (MHC) class II gene DRB locus in three feline species were examined on clouded leopard ( Neofelis nebulosa), leopard ( Panthera pardus) and Amur tiger ( Panthera tigris altaica). A pair of degenerated primers was used to amplify DRB locus covering almost the whole exon 2. Exon 2 encodes the β1 domain which is the most variable fragments of the MHC class II molecule. Single-strand conformational polymorphism (SSCP) analysis was applied to detect different MHC class II DRB haplotypes. Fifteen recombinant plasmids for each individual were screened out, isolated, purified and sequenced finally. Totally eight distinct haplotypes of exon 2 were obtained in four individuals. Within 237 bp nucleotide sequences from four samples, 30 variable positions were found, and 21 putative peptide-binding positions were disclosed in 79 amino acid residues. The ratio of nonsynonymous substitutions ( d N ) was much higher than that of synonymous substitutions ( d S ), which indicated that balancing selection probably maintain the variation of exon 2. MEGA neighbor joining (NJ) and PAUP maximum parsimony (MP) methods were used to reconstruct phylogenetic trees among species, respectively. Results displayed a more close relationship between leopard and tiger; however, clouded leopard has a comparatively distant relationship form the other two. [ABSTRACT FROM AUTHOR]

A non‐sense mutation in either DPA1*01:03:01:02 or DPA1*01:03:01:05/01:03:01:15 results in the novel allele, HLA‐DPA1*01:35N. [ABSTRACT FROM AUTHOR]

Potential links between fish life history traits, an immune investment as measured by variability of the MHC genes and parasitism were analysed in 14 species of cyprinid fish. The hypothesis of the diversity of MHC genes being driven by high parasite diversity, i.e. species richness, was tested and a potential relationship between the MHC diversity and fish life-history traits including adult mortality rate, fecundity, longevity and maturity was investigated. Molecular techniques (SSCP and sequencing) were applied to analyse the MHC nucleotide diversity of the exons 2 and 3 of DAB genes belonging to the MHC class IIB. The comparative analyses, using phylogenetic independent contrasts, revealed a negative relationship between parasite species richness and adult fish mortality rate. We also found a positive relationship between nucleotide diversity of the exon 2 and parasite species richness. Our results suggest that fish species, of which populations are exposed to high parasite pressure, in terms of high parasite species richness, maintain a high genetic diversity of the exon 2 of the MHC genes (presenting the peptide binding regions), allowing them to decrease their natural mortality rate. [ABSTRACT FROM AUTHOR]

Conclusions. In the samples of squamous cell carcinoma (SCC) of the head and neck region studied, mutations of exon 2 were quite rare. In contrast to the other three mutations, the G to C transversion on position 169 (Ala57Pro) has been found in several other studies before. Further investigations on mutations of the other exons 1α, 1β and 3 are necessary. Objective. Alterations of p16INK4A have been found in different types of human cancer cell lines. The aim of this study was to perform a screening for alterations of exon 2 of different SCCs of the head and neck region. Methods. Forty samples of different SCCs of the head and neck were collected by biopsy or surgical resection. Exon 2 was amplified by using the primers 5′-GGTGAGGGGGCTCTACACAAG-3′ and 5′-GTCTCCCGGGCTGAACTTTC-3′. DNA was sequenced with the primer 5′-CGGGCTGAACTTTCTGTGCT-3′. Results. Mutations were found in 4 of our 40 samples. One sample showed a G to C transversion on position 169 (Ala57Pro), a second sample from the same patient confirmed the latter transition but also contained a transition C to T on position 179 (Ala60Val). Another patient showed an already known polymorphism, a transition G to A at position 442 (Ala148Thr). The third case was a transversion C to A on position 213 (Asn71Lys). [ABSTRACT FROM AUTHOR]

Ovar-DRB1 is part of the major histocompatibility complex (MHC) class II of sheep and functions by presenting extracellular-derived peptides to the immune system. Although there are over 100 different Ovar-DRB1 DNA sequences reported in GenBank, only two Ovar-DRB1 mRNA sequences have been reported. As a first step in understanding MHC Class II function as it relates to disease progression in sheep, Ovar-DRB1 transcripts encoding the peptide-binding site or the first domain (β1) of Ovar-DRB1 in a 32-ewe–lamb flock were identified and characterized by using reverse transcriptase–polymerase chain reaction, cloning, sequencing, and phylogenetic analysis. Fourteen new Ovar-DRB1 β1 cDNA sequences out of a total of 15 Ovar-DRB1 β1 cDNA sequences in a ewe–lamb flock of 32 sheep were identified. One Ovar-DRB1 β1 cDNA sequence was 100% identical to M93432, one of the two Ovar-DRB1 mRNA sequences reported in GenBank. Twelve out of 15 Ovar-DRB1 β1 cDNA sequences were 100% identical to the corresponding previously reported Ovar-DRB1 genomic DNA sequences, indicating that these Ovar-DRB1 genomic DNA sequences are also transcribed. One of three of the remaining Ovar-DRB1 β1 cDNA sequences, DRB1*07012, had a synonymous substitution resulting in an identical deduced amino acid sequence to DRB1*0701. Two of the remaining three Ovar-DRB1 β1 cDNA sequences had nucleotide differences and subsequent deduced amino acid sequence differences when compared to known Ovar-DRB1 β1 genomic DNA sequences, and therefore, DRB1*0206 and DRB1*0353 represent new Ovar-DRB1 β1 expressed alleles. Phylogenetic analysis of the 15 Ovar-DRB1 β1 cDNA sequences revealed that DRB1*0206 had a strong phylogenetic relationship to DRB1*0203, and DRB1*0353 had a strong phylogenetic relationship to DRB1*0303. [ABSTRACT FROM AUTHOR]

Stickler syndrome (progressive arthro-ophthalmopathy) is a genetically heterogeneous disorder resulting from mutations in at least three collagen genes. The most common disease-causing gene is COL2A1, a 54-exon–containing gene coding for type II collagen. At least 17 different mutations causing Stickler syndrome have been reported in this gene. Phenotypically, it is also a variably expressed disorder in which most patients present with a wide range of eye and extraocular manifestations including auditory, skeletal, and orofacial manifestations. Some patients, however, present without clinically apparent systemic findings. This observation has led to difficulty distinguishing this Stickler phenotype from other hereditary vitreoretinal degenerations, such as Wagner syndrome and Snowflake vitreoretinal degeneration. In this regard, review of the literature indicates type II collagen exists in two forms resulting from alternative splicing of exon 2 of the COL2A1 gene. One form, designated as type IIB (short form), is preferentially expressed in adult cartilage tissue. The other form, designated as type IIA (long form), is preferentially expressed in the vitreous body of the eye. Because of this selective tissue expression, mutations in exon 2 of the COL2A1 gene have been hypothesized to produce this Stickler syndrome phenotype with minimal or absent extraocular findings. We review the evidence for families with exon 2 mutations of the collagen COL2A1 gene presenting in a distinct manner from families with mutations in the remaining 53 exons, as well as other hereditary vitreoretinal degenerations without significant systemic manifestations. [Copyright &y& Elsevier]

To determine the clinical characteristics and genetic causes of Waardenburg syndrome type 1 (WS1) present in a Chinese Han family. Evaluations, including the familial history, clinical features and audiological tests, were performed on the proband and her parents. Genetic analyses were conducted using targeted next-generation sequencing of 144 known deafness genes, and confirmed by Sanger sequencing. Bioinformatics analyses of the candidate variant were performed. The proband suffered from moderate hearing loss of the right ear, and her mother suffered from profound congenital bilateral hearing loss. The proband exhibited a left blue iris. The calculated W index of the proband was 2.61, while her mother's W index was 2.12. The proband and her mother were diagnosed with WS1 according to the Waardenburg Syndrome Consortium criteria. A novel missense variant NM_181457.3: c.127G > T; p.(Gly43Cys) in exon 2 in Paired Box 3 (PAX3) was identified in the proband and her mother, but this variant was not detected in the father and the controls. This variant was not reported in the HGMD, ClinVar, 1000G and ESP6500 databases. We identified a novel missense variant in exon 2 of PAX3 as the genetic cause of WS1 in this two-generation family, which broadened the genetic spectrum of WS1. [ABSTRACT FROM AUTHOR]

Major histocompatibility complex (MHC) genes are often implicated in disease resistance, sexual selection and local adaptation in salmonids, a highly studied and socio-economically important taxa. However, genotyping highly polymorphic genes is difficult, expensive, and prone to PCR and cloning artifacts that can result in false alleles. With the advent of next generation sequencing, it is possible to effectively 'clone' PCR products in a massively parallel fashion with the use of individually-tagged fusion primers. Primers that amplify a gene of interest across a variety of taxa, a single set of 50-150 primers can facilitate cost efficient genotyping and become a common lab resource. Here we developed MHC IIβ exon 2 primers for full-length amplification, including an additional PBR region, and demonstrate effectiveness with representatives from five genera of salmonids. These primers may facilitate parallel next generation sequencing for efficient, cost effective, and accurate genotyping of this complex locus. [ABSTRACT FROM AUTHOR]

Background: Rat Sarcoma (RAS) protein encoded Guanosine Triphosphate (GTP-ase) activity, known as a switch of cell proliferation. The mutation of this protein alters the early stage of carcinogenesis and along with the interaction with other oncogene drivers and environmental factors affect the clinical characteristics and prognosis in cancer patients, particularly lung cancer., Objective: This study aims to determine the Kristen Rat Sarcoma (KRAS) mutation in lung cancer patients in North Sumatera and evaluate factors that might contribute in the development of lung cancer in the absence of KRAS mutation., Methods: This was a retrospective cohort study enrolled 44 subjects age > 18 year with the diagnosis of lung cancer. Histopathology preparation was obtained from surgery, bronchoscopy, and percutaneus needle biopsy then formed as paraffin-block. KRAS mutation was analyzed using Polymerase Chain Reaction (PCR) method with specific primer of exon 2 for evaluating the expression of RAS protein then continued with Sanger Sequencing Method at 12th and 13th codon., Results: The majority of subjects were male, age > 40 years old, bataknese, heavy smoker, with Adenocarcinoma. Almost all the subjects showed the expression of exon 2 of RAS protein in PCR examinations. However, Sequencing analysis using Bioedit Software, BLASTs and Finch T showed GGT GGC as protein base 219-224 which represented 12th and 13th Codon 12 and 13. The results interpreted there was no mutations of exon 2 of KRAS in North Sumatera Population., Conclusion: The absence of KRAS mutation in exon 2 in several ethnics in North Sumatera populations was not the main factors of lung cancer., Competing Interests: There are no conflicts of interest., (© 2021 Noni Novisari Soeroso, Fannie Rizki Ananda, Andika Pradana, Setia Putra Tarigan, Elisna Syahruddin, Dimas Ramadhian Noor.)

In order to isolate, clone, and sequence agouti exon 2 of the pig (Yorkshire), we used an interspecific hybridization strategy. Primers from the 5′ and 3′ borders of the known human agouti exon 2 sequence were used to amplify (PCR) pig agouti exon 2. Following Southern blotting using a human exon 2 internal primer to authenticate that our PCR amplified product was truly pig exon 2 (PorAex2), the fragment was cloned and sequenced. PorAex2 exhibits 79.1 and 75.7% DNA sequence and 85 and 74% deduced amino acid sequence homologies with human and mouse agouti exon 2 and agouti protein, respectively. With the isolation of PorAex2, we can now map, sequence, and clarify the modus operandi of the porcine agouti gene. The GenBank Accession number of PorAex 2 is AF018166. [ABSTRACT FROM AUTHOR]

The article presents verification results of novel DRB1 1520 allele in human leucocyte antigen. The Exon 2 sequence analysis is used to study HLA-DRB1 allele, DRB1 1520. Cloning and gene sequence is used to verify the presence of the allele. The article reports the genomic DNA was extracted from peripheral blood mononuclear cells using the QIAamp Blood Kit and allele-specific amplification for complete exon 2. The results shows DRB1 1520 contains sequence motifs of DRB1 150101 allele.

Presents the exon 2 sequence analysis of a novel HLA-DRB1 allele called DRB1*1450. Extraction of genomic DNA from peripheral blood mononuclear cells using the QIAamp Blood Kit; Isolation of plasmid DNA using the QIAprep Spin Miniprep Kit; Alignment of sense and antisense sequences.

We describe the identification of a new DRB1*03 variant in a Caucasian volunteer bone-marrow donor (DR’KW’). The indication for the existence of this new variant arose from contradictory results obtained by serological and several DNA typing methods (SSP, SSOP). Analysis of the exon 2 sequence revealed that the DRB1*0314 contained the characteristic DR3 specific sequence motifs, except for codon 77 (AAC→ACC) where threonine is substituted for asparagine. [ABSTRACT FROM AUTHOR]