Sanger Sequencing Technique Research Articles

Detection of CYP21A2 gene mutations and the differences in the levels of hormones in patients with 21-hydroxylase deficiency

Objective: To analyze the detection of CYP21A2 gene mutations in 21-hydroxylase deficiency (21-OHD) patients, so as to determine the accuracy of clinical diagnosis. Methods: Totally, 514 patients with 21-OHD who visited Peking Union Medical College Hospital from January 2015 to January 2018 were enrolled and their clinical and biochemical data were collected. DNAs were extracted from peripheral blood leukocytes and CYP21A2 mutations were detected by Sanger sequencing and multiple ligation probe amplification (MLPA) technique. We divided 514 patients into three groups: two mutations of CYP21A2 alleles (group A), one mutation of CYP21A2 (group B), and no mutation of CYP21A2 (group C). Results: Mutation was detected in each allele of CYP21A2 gene in 401 (78.0%) patients, ninety (17.5%) had only one mutant allele and 23 (4.5%) had no mutation. There was no significant difference between the patients with different clinical phenotypes and the number of CYP21A2 gene mutations detected. In male, the cortisol of the patients with simple virilizing 21-OHD in group A [0.04 (0.02, 0.20) nmol/L] was lower than that of group B [0.24 (0.17, 0.28) nmol/L] and the difference was statistically significant (P=0.014). In female, 17-hydroxyprogesterone (17-OHP) of patients with salt wasting 21-OHD in group A [153.7 (90.1, 204.5) nmol/L] was higher than that of group B [38.2 (31.0, 183.3) nmol/L] and C [42.6 (27.8, 48.1) nmol/L] and the differences were statistically significant (both P<0.05). The progesterone of patients with simple virilizing 21-OHD in group C [23.0 (8.6, 33.2) nmol/L] was lower than that of gourp A [57.8 (34.4, 110.2) nmol/L] and B [63.6 (31.4, 110.8) nmol/L] and the difference were statistically significant (both P<0.05). The 17-OHP of patients with non-classical 21-OHD in group C [24.5 (20.4, 54.2) nmol/L] was lower than that of group A [158.7 (59.1, 187.6) nmol/L] and B [147.8 (131.9, 179.3) nmol/L]. The difference were statistically significant (both P<0.05). Conclusions: Mutations of two alleles have not been found in all patients with clinically diagnosed 21-OHD. Other congenital adrenal hyperplasia (CAH) types which can cause similar changes in 17-OHP and other hormones may be misdiagnosed as 21-OHD. Therefore, 21-OHD cannot be diagnosed with help of 17-OHP level only, and gene detection plays a vital role in the differential diagnosis of different CAH types.

A report of Hb Fontainebleau [α21 (B2) Ala > Pro] as a result of founder effect phenomenon

ObjectiveTo date, more than 400 alpha chain variants or point mutations in the α-globin genes that lead to single amino acid substitutions have been described. Here, we state a rare alpha-globin gene variant Hb Fontainebleau [a21 (B2) Ala>Pro] identified in heterozygote state in two families from the north of Iran during premarital thalassemia screening program. MaterialTwo first cousin couples with reduced hematological indices compatible with α-thalassemia were referred to Lab for premarital screening of thalassemia. At first CBC and capillary electrophoresis tests were applied. For molecular investigation genomic DNA was isolated from peripheral blood samples. Then, multiplex-Gap PCR and Sanger sequencing techniques were applied to discover common α- globin deletions and probable point mutations, respectively. ResultsIn the capillary electrophoresis results, one unknown peak was observed to the left of Hb A. Sanger sequencing results revealed that all cases are heterozygote for Hb Fontainebleau (HBA2: c.64G4C, p.Ala21Pro). Family history analysis has shown that all presented cases have belonged to the families which are originally from India. ConclusionThe presence of Hb Fontainebleau in Mazandaran province and in subjects with Indian origin may indicate the founder effect phenomenon. DNA analysis of subjects with reduced hematological indices is recommended for finding common and rare mutations to predict childbirth with thalassemia in premarital screening programs.

Genetic analysis of familial hypercholesterolemia in Asian Indians: A single-center study

Familial hypercholesterolemia (FH), an autosomal codominant disorder characterized by very high low-density lipoprotein cholesterol, is strongly associated with premature coronary artery disease. Molecular landscape of FH in Asian Indians is not well studied, although this ethnic group comprises a large proportion of the world population. Knowledge of mutations in these groups is useful for identifying persons affected with FH, saving their lives, and cascade screening in their relatives. Potential cases of FH (n=100) were identified by criteria adapted for the Indian population from Dutch Lipid Clinic Network criteria. Pathogenic variants were analyzed in LDLR, APOB 100 (exons 26 and 29), PCSK9, and APOE genes using Sanger sequencing and multiplex ligation-dependent probe amplification technique. Cases in whom there were no pathogenic variants were tested by next-generation sequencing using a targeted panel of genes. Thirty-eight pathogenic variants were identified in 47 of 100 unrelated probands. Of these variants, 33 were identified in LDLR, 3 in APOB, and 2 in PCSK9 genes. Ten pathogenic variants were novel. Mutations were detected in 91.4% of those subjects classified as definite, 40% as probable, and in 18.8% as possible FH cases based on modified Dutch Lipid Clinic Network criteria. A likely founder mutation in intron 10 (c.1587-1G>A) of LDLR gene was observed in 6 North Indian families. The conventional pathogenic variants in APOB and PCSK9 genes and those previously reported in LDLR gene among Asian Indians were not detected in this cohort. This study demonstrates genetic heterogeneity of FH in India. The variants observed were different from those described in Western populations. Next-generation sequencing technology helped identify new mutations in APOB gene, suggesting that in less-studied populations, it is better to sequence the whole gene rather than test for specific mutations.

Deciphering the Causal Diagnosis of Hydrops Fetalis or Unexplained Fetal Anemia Using Targeted Next Generation and Exome Sequencing

Fetal anemias are serious complications during pregnancies, which could lead to fetal death in case of hydrops fetalis (1/3000 pregnancies). Fetal anemia and hydrops fetalis are in most cases the result of fetal maternal alloimmunization, parvovirus B19 infection, fetal maternal hemorrhage, chromosomal abnormalities, congenital malformations, metabolic diseases, and in the context of hematological disorders, alpha-thalassemia. However, in one case out of 5, fetal anemia remains unexplained after an exhaustive first line of etiological evaluation. In order to identify the cause of the unexplained fetal anemia and to provide advice regarding prenatal diagnosis for next pregnancy, we have developed useful diagnostic tools on fetal blood based on erythrocyte and reticulocyte indices, examination of red cell morphology, flow cytometry (EMA test), osmotic gradient ektacytometry and molecular screening analysis. 43 fetal samples (30 probands) have been referred to our Hematology diagnostic lab in the time span between 2012-2018. In majority of the cases, various analyses were performed on fetal blood (23 out of 43) and in other instances during post-mortem examination following death (17 out of 43). Fetal blood purity was confirmed by microsatellite analysis on both parental and fetal DNAs. Informed consent was obtained from the mother in all cases. In 6 of 43 cases, prenatal diagnosis was performed after identification of the causal mutation responsible for the hydrops fetalis in the first fetus. Hydrops fetalis was suspected in 24 cases at the time of fetal sample collection. Red cell morphology and ektacytometry enabled the establishment of clinical diagnostic in two cases (congenital dyserythropoiesis type II (CDAII) and xerocytosis). Molecular screening analysis was performed by Sanger sequencing technique from 2012 to July 2016 and subsequently we designed a targeted Next Generation Sequencing (NGS) library including 74 genes involved in red cell disorders (n=9 fetuses) and exome sequencing (WES) was performed for 4 fetuses. Each identified allelic variation was confirmed by Sanger sequencing technique. Molecular Biology analysis (except the 6 prenatal diagnosis cases) was performed on 21 of 37 fetuses that enabled identification of the molecular defect in 10 fetuses. Rare red cell disorders were diagnosed in these cases including Diamond-Blackfan anemia (n=2), congenital dyserythropoiesis (n=6) and stomatocytosis (n=2) respectively. No putative pathogenous allelic variation following molecular screening could be identified in 4 fetuses. In the 6 cases which were screened for the molecular defect previously identified, none of the tested fetuses exhibited the allelic variation identified in the first fetus. In summary, targeted-NGS and WES are very valuable tools in the causal diagnosis of hydrops fetalis dues to unexplained anemia in addition to routine hematological tests (erythrocyte and reticulocyte indices, red cell morphology, flow cytometry, and ektacytometry) and after elimination of the most frequent causes of hydrops fetalis. This clinical problem is more frequent than has been previously surmised and needs more attention. Disclosures No relevant conflicts of interest to declare.

Evaluation of sanger sequencing method for ESR1 mutation detection in primary breast tumor

Objective: Exon 8 estrogen receptor 1 (ESR1) mutations in the ligand-binding domain play an important role in mechanisms of hormonal therapy resistance in breast cancer. Identification of ESR1 mutations is very important in determining the appropriate steps of therapy. In this study, we evaluate Sanger sequencing technique to detect the ESR1 mutation. Methods: This retrospective study was conducted using 49 advanced breast cancer patients with estrogen receptor positive. Deoxyribonucleic acid (DNA) material was extracted from primary breast tumor samples. Exon 8 ESR1 gene mutation was analyzed by Sanger sequencing method using BigDye Direct Sequencing Kit (Applied Biosystem) with gBlock synthesis gene fragment (Integrated DNA Technologies) D538G as a positive control. Results: The mean age of patients was 46.14 (±9.6) years, and 61.2% were in stage 4. There are no exon 8 ESR1 gene mutations detected in 49 primary tumor samples, whereas the gBlock-positive control showed base substitution in 1613A>G (D538G) indicating the success of sequencing reaction. Conclusion: Sanger sequencing has failed to detect ESR1 mutation in primary tumor breast samples. Other advanced molecular techniques should be performed for diagnosis of primary breast tumors.

Genomic evidence suggests Mesapamea remmi is an imaginary species (Lepidoptera: Noctuidae)

AbstractTaxonomic status of the noctuid moth Mesapamea remmi Rezbanyai‐Reser (Lepidoptera) has remained controversial since its description in 1985. Based on morphology, it has been considered a valid species or a hybrid between Mesapamea secalis (Linnaeus) and Mesapamea didyma (Esper). We studied this case of uncertain identity by using traditional Sanger sequencing techniques (COI and seven nuclear genes) as well as double digest restriction‐site associated DNA (ddRAD) sequencing, which resulted in analyses including 9402 loci and 1 512 976 bp across the genome. Our analyses showed that genomic data do not support the hybrid hypothesis and that M. remmi is not separated genetically from M. secalis; both are clearly distinct from M. didyma. Reproductive organs of M. remmi males are unique and diagnosable, whereas females are teratological in the sense that ductus seminalis is missing and corpus bursae is malformed, the latter being connected directly to the ovipore. These data support the view that M. remmi is not a valid species and we consider M. remmi Rezbanyai‐Reser, 1985 to be a junior synonym of M. secalis (Linnaeus, 1758). We demonstrate that genomic approaches provide an efficient way to address various difficult and long‐standing taxonomic issues, in this case oversplitting of species.

The influence of a genetic variant in the KCNQ1 gene on type 2 diabetes mellitus development

BackgroundType 2 diabetes mellitus (T2DM) is a complex metabolic disorder caused by environmental and genetic factors. PurposeHere, we provided the evidence for the association of rs2237895 variant of potassium voltage-gated channel subfamily Q member 1 (KCNQ1) gene with the risk of T2DM in a group of Iranian patients. MethodsKCNQ1 single nucleotide polymorphism (SNP) genotypes were characterized in a representative sample of 97 patients with T2DM and 97 healthy volunteers using Sanger sequencing technique. ResultsWe observed that T2DM patients did not significantly differ with healthy individuals in genotypes and alleles frequency of rs2237895 KCNQ1 gene (P = 0.7 and P = 0.6, respectively). Frequencies of genotypes of rs2237895 polymorphism (AC + CC) among T2DM patients with renal complication were significantly lower than T2DM patients without (42.86% and 91.67%, respectively, P = 0.038, OR = 0.07, and 95% CI = 0.005–0.86). ConclusionOur result showed that rs2237895 KCNQ1 gene polymorphism was not associated with T2DM but the presence of this variant significantly prevented developing renal complications.

Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing.

Background:The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious obligate anaerobic bacteria are mostly associated with abscesses in animals. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing.Aim:This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals.Materials and Methods:This study performed a pilot investigation of abscesses from 20 clinical cases (17 rabbits, 2 hedgehogs, and 1 sugar glider) using standard culture methods for both aerobes and anaerobes and broad-range nested PCR targeting the 16S rRNA gene followed by the Sanger sequencing technique.Results:The standard culture and PCR techniques detected bacteria in 9 and 17 of 20 samples, respectively. From the 17 sequencings of the 16S rRNA, 10 PCR products were found to be closely related with obligate anaerobes including Bacteroides spp., Fusobacterium spp., Prevotella spp. Phylogenetic analysis using the rpoB gene revealed that the species for the Bacteroides was thetaiotaomicron and for the Fusobacterium was varium and nucleatum. However, the amplification of the rpoB gene for the Prevotella spp. was unsuccessful. Correlations between the standard culture and PCR techniques were found in 9 (6 positive and 3 negative samples) of 20 samples. Eleven samples were discordant between the standard culture and PCR techniques which were composed of eight samples negative by culture but positive by PCR and three samples had different bacteria by the culture and PCR techniques.Conclusion:According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture.

Multimodal Imaging and Functional Testing in a North Carolina Macular Disease Family: Toxoplasmosis, Fovea Plana, and Torpedo Maculopathy Are Phenocopies

To describe multimodal imaging and corresponding functional studies in a newly found family with North Carolina macular dystrophy (NCMD). To our knowledge, this is an original report using OCT angiography to evaluate persons with NCMD. A descriptive, retrospective study of a family with NCMD. A total of 3 participants, representing 3 generations of a single family, each demonstrating a different grade of NCMD, underwent clinical and genetic testing. Diagnostic multimodal imaging and functional testing of the retina included color fundus photography, fundus autofluorescence, intravenous fluorescein angiography, spectral-domain OCT and OCT angiography, multifocal electroretinography, full-field electroretinography, and microperimetry. DNA sequencing was performed using Sanger sequencing techniques. Spectral-domain OCT images, fundus photographs, fundus autofluorescence images, fluorescein angiograms, OCT angiograms, multifocal electroretinography images, full-field electroretinography images, and microperimetry maps. Sanger sequencing was performed for molecular diagnosis. Multimodal imaging helped to demonstrate the nature of the retinal and choroidal lesions in each participant and the extent of visual function. Genetic testing demonstrated the variant 2 point mutation (chromosome 6: 99593111) in the deoxyribonuclease 1 hypersensitivity binding site on chromosome 6q16 causing overexpression of the retinal transcription factor PRDM13. NCMD has great phenotypic variability, which can be appreciated only by examining multiple family members. To our knowledge, this is an original report that shows a correlation of functional studies with multimodal imaging. These findings are consistent with NCMD being a developmental abnormality of the macula. All layers of the retina and choroid demonstrate maldevelopment and varying degrees of malfunction. Although PRDM13 is expressed in the amacrine cells, we have yet to identify an abnormality specific to this cellular layer. The retinal vasculature appears to be surprisingly well preserved or intact by OCT angiogram compared with that shown in intravenous fluorescein angiograms. OCT angiograms suggest that foveal hypoplasia is a phenocopy of grade 1 NCMD, torpedo maculopathy is a phenocopy of grade 2 NCMD, and in this single family, congenital toxoplasmosis is a phenocopy of grade 3 NCMD.

Correlations of IL-17 and NF-κB gene polymorphisms with susceptibility and prognosis in acute respiratory distress syndrome in a chinese population.

The present study was performed to investigate the association between interleukin-17 (IL-17) and nuclear factor κB (NF-κB) gene polymorphisms and the risk and prognosis of acute respiratory distress syndrome (ARDS) in a Chinese population. A total of 210 Chinese patients with ARDS were selected as the study group, 210 individuals who were identified as at-risk patients but did not meet criteria for ARDS were recruited as the control group. Three single nucleotide polymorphisms (SNPs) of IL-17, including rs763780 (A>G), rs2275913 (G>A), rs8193036 (C>T) and NF-κB1 gene rs3774934 (G>A) loci were examined by Sanger sequencing technique in the peripheral blood of all subjects. Patients were followed for 30-day survival. The IL-17 rs763780 and NF-κB1 rs3774934 SNPs had no impact on ARDS risk and prognosis of ARDS (P>0.05). Compared with individuals carrying the wild-type GG genotype of rs2275913 at IL-17, the AA-homozygous and GA- heterozygous individuals were protected from the development of ARDS. Consistently, a decreased 30-day mortality risk was found among A-allele carriers of rs2275913 at IL-17 (p<0.05). For IL-17 rs8193036 SNP, the homozygote TT genotype and heterozygote CT genotypes were associated with increased ARDS susceptibility and 30-day mortality risk (P<0.05). Besides, decreased IL-17 levels were found in A-allele carriers of IL-17 rs2275913, whereas individuals carrying T-allele of IL-17 rs8193036 were found to have significantly increased levels of IL-17 (P<0.05). Our results suggested that two functional polymorphisms of IL-17, rs2275913 and rs8193036 were associated with ARDS risk and prognosis, indicating that the two genetic variants might act as possible markers for the prediction of ARDS risk and development.

Complete mitochondrial genome of Stichaeus nozawae Jordan & Snyder 1902 (Zoarcales: Stichaeidae)

We report on the structure and composition of the first complete mitochondrial genome of Stichaeus nozawae (typical prickleback fish (Zoarcales, Stichaeidae)), obtained by dideoxy (Sanger) sequencing technique. Genome has a classic organization with 13 genes, 2 rRNAs, control region, and 22 tRNAs, including additional tRNAs with atypical codons previously found in many eelpouts. Phylogenetic analysis confirms the species identity of S. nozawae and validate its recent divergence from S. grigorjewi with unusually low interspecific genetic distance.

Modeling early genomic events of pancreatic cancer using CRISPR/Cas9

Background: Duct epithelial cells transform into pancreatic cancer based on specific genetic alterations. Regards the lack of untransformed duct epithelial cell lines, no conclusive in vitro carcinogenesis model exists. A sensitive immortalization strategy (hTERT) was used to establish a new duct epithelial cell line. Subsequently CRISPR/Cas9 genome editing was applied modeling an early genomic event of pancreatic cancer. Methods: Non-neoplastic pancreatic ducts were isolated and immortalized using a lentiviral transfer-vector containing the hTERT gene. IHC and FISH analyses assessed telomerase expression. Growth rate and cellular kinetics were monitored. Single guide(sg)RNA close to target region were sub-cloned into Cas9n plasmid constructs. These constructs were transfected together with KRASG12D homology donor. FACS sorted clones are screened using Sanger sequencing technique. Results: Lentiviral transduction resulted in hTERT expressing cells. After selection, cytokeratin expressing duct cells amplified in a cobblestone pattern with robust cellular kinetics calling for a new Primary Duct Cell Line (PDCL). IHC- and FISH-analysis indicated sustained telomerase activity. Cells were successfully transfected with sgRNA and homology donor, targeting KRASG12D. Following FACS sorting, monoclonal cells were screened for KRASG12D clones. Conclusion: We successfully introduced KRASG12D sgRNA constructs into a new PDCL. This might be the baseline for modeling tumorigenesis in vitro of pancreatic cancer.

Modeling early genomic events of pancreatic cancer using CRISPR/Cas9

Background: Duct epithelial cells transform into pancreatic cancer based on specific genetic alterations. Regards the lack of untransformed duct epithelial cell lines, no conclusive in vitro carcinogenesis model exists. A sensitive immortalization strategy (hTERT) was used to establish a new duct epithelial cell line. Subsequently CRISPR/Cas9 genome editing was applied modeling an early genomic event of pancreatic cancer. Methods: Non-neoplastic pancreatic ducts were isolated and immortalized using a lentiviral transfer-vector containing the hTERT gene. IHC and FISH analyses assessed telomerase expression. Growth rate and cellular kinetics were monitored. Single guide(sg)RNA close to target region were sub-cloned into Cas9n plasmid constructs. These constructs were transfected together with KRASG12D homology donor. FACS sorted clones are screened using Sanger sequencing technique. Results: Lentiviral transduction resulted in hTERT expressing cells. After selection, cytokeratin expressing duct cells amplified in a cobblestone pattern with robust cellular kinetics calling for a new Primary Duct Cell Line (PDCL). IHC- and FISH-analysis indicated sustained telomerase activity. Cells were successfully transfected with sgRNA and homology donor, targeting KRASG12D. Following FACS sorting, monoclonal cells were screened for KRASG12D clones. Conclusion: We successfully introduced KRASG12D sgRNA constructs into a new PDCL. This might be the baseline for modeling tumorigenesis in vitro of pancreatic cancer.

Ultra-Deep Sequencing Leads to Earlier and More Sensitive Detection of Known or Novel Mutations of BCR-ABL1 in Chronic Myeloid Leukemia

Detection of BCR-ABL1 mutations that inhibit tyrosine kinase inhibitors is important for the treatment of CML patients. Sanger sequencing (SS) technique is considered the gold standard for mutation detection in a clinical laboratory. We analyzed 40 clinical samples from 22 CML patients with TKI resistance by ultra-deep sequencing (UDS) and compared the results with classical SS-based tests. UDS facilitates the detection of low levels of mutations (<20% allele frequencies) that SS does not detect. In subgroups of cases with multiple mutations, UDS was also able to determine whether two mutations were on same BCR-ABL1 allele (compound) or not (polyclonal). We found a pair of compound mutations unreported: F359C/E450 which confer high-level resistance to first-generation TKI imatinib, second-generation TKIs dasatinib and nilotinib, third-generation TKI ponatinib and a new TKI GZD824. UDS provides a reliable method for identifying low levels of BCR-ABL1 mutations. DisclosuresNo relevant conflicts of interest to declare.

Development of an AmpliSeqTM Panel for Next-Generation Sequencing of a Set of Genetic Predictors of Persisting Pain

Background: Many gene variants modulate the individual perception of pain and possibly also its persistence. The limited selection of single functional variants is increasingly being replaced by analyses of the full coding and regulatory sequences of pain-relevant genes accessible by means of next generation sequencing (NGS).Methods: An NGS panel was created for a set of 77 human genes selected following different lines of evidence supporting their role in persisting pain. To address the role of these candidate genes, we established a sequencing assay based on a custom AmpliSeqTM panel to assess the exomic sequences in 72 subjects of Caucasian ethnicity. To identify the systems biology of the genes, the biological functions associated with these genes were assessed by means of a computational over-representation analysis.Results: Sequencing generated a median of 2.85 ⋅ 106 reads per run with a mean depth close to 200 reads, mean read length of 205 called bases and an average chip loading of 71%. A total of 3,185 genetic variants were called. A computational functional genomics analysis indicated that the proposed NGS gene panel covers biological processes identified previously as characterizing the functional genomics of persisting pain.Conclusion: Results of the NGS assay suggested that the produced nucleotide sequences are comparable to those earned with the classical Sanger sequencing technique. The assay is applicable for small to large-scale experimental setups to target the accessing of information about any nucleotide within the addressed genes in a study cohort.

DUCHENNE MUSCULAR DYSTROPHY - GENETICS: P.214Genetic profile of Chilean patients with Duchenne muscle dystrophy

Duchenne muscle dystrophy (DMD), is a progressive muscular disease, inherited as a X link. Is caused by the mutations of the dystrophin gene. Deletions of one or more exons account for approximately 50-65%, duplications 5-10% and point mutations 20-25%. Analyze the genetic profile of patients with DMD through regular checkups at the pediatric neurology department of the San Borja Arriarán Hospital. Retrospective descriptive study. Analysis of clinical records of 41 patients in evaluation by DMD, excluding patients diagnosed prior to the year 2010, with only biopsy and/ or PCR (polymerase chain reaction) without alterations. 41 patients with clinical manifestations and/or muscle biopsy with markedly reduced or no detectable dystrophin immunostaining, were studied by PCR, MLPA (multiplex ligation probe amplification) and/or Sanger sequencing techniques. 27/41 (66%) of patients had large deletions, 20/27 had compromised exons 44-55, and 3/26 had compromised exons 3-7. The most frequent deletion was exon 50, followed by exon 49. 3/41 (7,3%) presented duplications, which all affected exon 2. 10/41(24,4%) of patients had a negative MLPA, but a compatible muscle biopsy. In 1/41, sequencing was performed, showing nonsense mutation, in exon 41. Our study of the genetic profile of chileans patients is similar to previously reported, deletions are the most frequent alterations. It should be noted that biopsy continues to be an important tool for confirming diagnostics, due to our countries limited access to sequencing techniques. Because of this, we can extrapolate and conclude that the large percentage of patients with compatible muscle biopsy and negative MLPA, could correspond to point mutations, which would then coincide with the published. Therefore, it is important to make genetic diagnosis advances thus giving us the possibility of integrating new therapies and the ability to offer genetic counseling.

Advantages and Drawbacks of Next Generation Sequencing

Starting in 1990 the Human Genome Project (HGP) allowed the sequencing of the whole human genome by 2003 (Gibson and Muse, 2009). The Sanger sequencing technique is a gold standard for sequencing DNA and was instrumental for HGP (Gibson and Muse, 2009). Unfortunately, Sanger sequencing involves much time and money to perform, and throughout the decades other methods of sequencing have been developed to reduce time and cost for sequencing large scale projects. In addition to time and cost of sequencing, hurdles have arisen — such as correct sequence assembly of repeated regions of DNA and how to navigate through mega-datasets. In this essay throughput, cost and other technological and biological aspects will be summarized for the next generation of sequencing technologies.

Macular and choroidal thickness of children with Familial Mediterranean Fever gene mutation

Introduction: The present study aimed to evaluate macular and choroidal thicknesses in children with MEFV gene mutations.Methods: Thirty-five children with MEFV gene mutations and 40 healthy controls were included in the study. MEFV gene profiles for the current cohort were genotyped by Pyrosequencing and direct Sanger sequencing techniques. The thicknesses of the macular and choroid of each subject’s right eye were measured using spectral-domain optic coherence tomography.Results: Mean arteriolar and venular diameters were 95.75±11.98 µm and 127.61±10.44 µm in children with the MEFV gene mutations and 110.19±11.10 µm and 138.54±10.04 µm in control group respectively. Children with the MEFV gene mutations were found to have a significantly less mean arteriolar diameter (p&amp;lt;0.001), mean venular diameter (p&amp;lt;0.001), less macular thickness (p=0.016), and less choroidal thickness (p=0.014) compared to healthy controls. Conclusion: Children with the MEFV gene mutations had narrowing of the retinal arterioles, retinal venules, and macular and choroidal thinning. Future studies should aim to investigate endothelial dysfunction in children with Familial Mediterranean Fever by non-invasive and effective methods.

A novel mutation in MERTK for rod-cone dystrophy in a North Indian family

ObjectiveTo identify the underlying genetic defect of childhood-onset severe rod-cone dystrophy (RCD) in a consanguineous family from North India with autosomal recessive retinitis pigmentosa. MethodsA detailed family history, clinical data, and blood samples were collected from 11 members of the family, including 4 affected by an autosomal recessive rod-cone dystrophy (arRCD), and DNA was extracted. Whole-exome sequencing (WES) was performed on DNA samples of proband and her unaffected maternal uncle. Ion Reporter software (ver. 4.4) was used for the annotation of variants obtained by WES. The variants detected in proband were tested for validation in all other affected and unaffected family members using Sanger sequencing technique. ResultsWe have identified a novel nonsense mutation—c.1647T>G (p.Tyr549Ter)—in the exon 11 of MERTK that co-segregated completely with the disease phenotype in all the 4 affected members and was not observed in the 7 unaffected members of the family. This mutation was also not detected in 120 ethnically matched controls (240 chromosomes), hence excluding it as a polymorphism. ConclusionsMERTK has a role in retinal pigment epithelium as a regulator of rod outer segments’ phagocytosis. Due to c.1647T > G substitution, the stop codon (p.Tyr549Ter) appears early in the transcript. It seems that either the altered transcript would degenerate through nonsense-mediated decay (NMD) or potentially form truncated protein lacking a functionally important domain (i.e., tyrosine kinase domain). These findings thus further expand the mutation spectrum in MERTK and substantiate its role in the pathogenesis of retinal dystrophy.

Cross sectional study to determine chloroquine resistance among Plasmodium falciparum clinical isolates from Khartoum, Sudan

Background: Malaria continues to present a global health threat; the World Health Organization (WHO) reported 214 million cases of malaria by the year 2015 with a death rate of 438000. Sudan is endemic to malaria with over 95% of malaria cases due to Plasmodium falciparum. Chloroquine is a well-established drug in the treatment of P. falciparum malaria although its use has declined since its introduction as the drug of choice in treatment of malaria in Sudan. The mechanism of resistance has been attributed to mutations in P. falciparum Chloroquine resistance transporter gene coding for a key food vacuole proteins. In current study we aimed at verifying the genetic cause of resistance to Chloroquine in field isolates of P. falciparum. Methods: Twenty P. falciparum cases were diagnosed from East Nile hospital in Khartoum and recruited in the investigation. Nested PCR was conducted to isolate mutation region in the PfCRT gene and the amplicons were sequenced using Sanger sequencing technique (Macrogen, Soule Korea). Results: 16/20 (80%) of the field isolates contained base pair mutation of codon 76 in the pfcrt gene thus being resistant to chloroquine treatment and only 4/20 (20%) did not contain such mutation. Conclusions: High treatment failures associated with Chloroquine treatment is evident of the high prevalence of mutant strains of P. falciparum field isolates thus suggesting the reduced relevance of Chloroquine as a treatment choice in the management of P. falciparum malaria.