Overview of Next-Generation Sequencing Technologies Slatko, Barton E; Gardner, Andrew F; Ausubel, Frederick M
Current protocols in molecular biology (Print),
04/2018, Volume:
122, Issue:
1
Journal Article
Megalencephalic leukoencephalopathy with subcortical cysts (MLC; MIM#604004) is a rare congenital disorder of the cerebral white matter. Most MLC patients have MLC1 mutations which are inherited in ...an autosomal recessive manner. Since there is no established biomarker for MLC, genetic testing is necessary for a final diagnosis. We identified compound heterozygous mutations in two different Japanese patients having MLC. Both patients showed a common mutation of c.278C>T (p.Ser93Leu) in a heterozygous status. In addition, two different mutations (c.337_353delinsG and c.423+1G>A) were identified in the homologous alleles of the patients, respectively. Although these two variants were first identified in the Japanese population, they have since been reported in other Asian countries and might be founder mutations of the gene in Asian population.
Introduction
Breast Cancer is the number one cause of cancer related deaths in Pakistani women with an incidence of 1 in 9. This is a part of a larger study that we conducted to identify the variants ...in 27 possible genes known for breast and ovarian cancer along with several oxidative stress markers. The current objective is to report a predictive novel variant of diagnostic importance in local females with breast cancer. Furthermore, to study the epidemiological and clinical presentation that may be predictive of this variable.
Methods
After ethical approval and informed consent, we extracted DNA from peripheral blood and conducted next generation sequencing followed by Sanger confirmation. Bioinformatic analysis was performed on ANNOVAR and SNP‐Nexus softwares.
Results
Our results on both softwares indicate that an identical novel Phosphatase and Tension Homolog (PTEN) variant is present in 69% patients. It is a homozygous frameshift substitution (GCGCCG > CCGCCGC) at amino acid position C65S on exon 2 of chromosome 10 from 89623901 to 89623906. It acts at Micro‐RNA and Cellular Senescence level of cancer pathways. All PTEN cases showed significant triple negative behavior at estrogen, progesterone and HER2 receptors, associated with adverse outcome. A total of 85% were premenopausal with mean age of 35 years +/‐ SD 5.611; SE 1.255; range 27‐46 years). Their mean age at menarche was 13.25 years +/‐ SD 0.716; SE 0.160. The mean BMI showed overweight status. Family history was positive and known in only 35% but they were mainly (60%) the outcome of related parents. In majority (75%) of them history of breast feeding was positive and had no difficulty in breast feeding (70%). Around 80% were non‐smokers and 100% were non‐alcoholics. Most of them (85%) presented with Invasive Ductal Carcinoma and 55% with grade III. They all were homemakers, most (85%) being married and majority (75%) did not receive education above grade 10. Nearly 70% used tap / filtered‐tap‐water for drinking. They all had unilateral breast cancer of right side mainly (60%). Almost all (95%) detected it by feeling the lump. Mastectomy was performed only in 30% and all of them underwent chemotherapy. Radiotherapy was offered to only 10%. A total of 35% among them had only 1 year post‐diagnosis survival.
Conclusion
This novel variant may be useful for researcher and clinician to expect similar outcome in unaffected carriers. It can be offered as a screening tool to achieve pre‐diagnosis around 13 years of age to predict similar clinical manifestation using affordable Sanger technique, even before the breast development. The early diagnosis may improve the outcome.
The taxonomic identification of species through analysis of the variation of orthologous DNA sequences, complement the information obtained with morphological characters. Cytogenetic studies indicate ...that polyploid taxa occur in the subfamily Opuntioideae, Opuntia ficus-indica, contributing to morphological variability in the individuals of a population, and influencing the correct identification of species. However, the lengths of the sequences in Opuntioideae are affected by the extraction of pure DNA. Different extraction methods were evaluated and modified, and a procedure was established to obtain good quality DNA, free of inhibitors for gene amplification by polymerase chain reaction. The ratio A260/A280 and A260/A230 ranged from 1.6 to 2.1, revealing absence of contamination with the modified protocol for DNA extraction from cotton leaves. This method is inexpensive compared to those of commercial manufacturers and, therefore, can be applied in studies with limited resources.
Introduction: In preceding work, a deleterious REEP4 variant GRCh38/hg38, NC_000008.11:g.22140245G>A, NM_025232.4:c.109C>T, p.Arg37Trp was found to co-segregate with blepharospasm (BSP) in a large ...African-American pedigree. Other REEP4 variants have been reported in genetic screening studies of dystonia. The REEP4 paralogs, REEP1 and REEP2 , are associated with spastic paraplegia. The causal contributions of REEP4 variants to dystonia and other neurological disorders remains indecisive. Methods: Sanger sequencing was used to screen subjects ( N = 307) with BSP and BSP-plus dystonia affecting additional anatomical segments (BSP+) phenotypes for variants in REEP4 . In silico tools were used to examine the deleteriousness of reported (ClinVar) and previously published REEP4 variants. Results: No highly deleterious variant was identified in coding or contiguous splice site regions of REEP4 in our cohort of 307 subjects. In silico analysis identified numerous deleterious REEP4 variants in published screening studies of dystonia and several highly deleterious single nucleotide REEP4 variants in ClinVar. Conclusion: Highly deleterious REEP4 variants are rare in BSP and BSP+ phenotypes.
Megalencephalic leukoencephalopathy with subcortical cysts (MLC; MIM#604004) is a rare congenital disorder of the cerebral white matter. Most MLC patients have MLC1 mutations which are inherited in ...an autosomal recessive manner. Since there is no established biomarker for MLC, genetic testing is necessary for a final diagnosis. We identified compound heterozygous mutations in two different Japanese patients having MLC. Both patients showed a common mutation of c.278C>T (p.Ser93Leu) in a heterozygous status. In addition, two different mutations (c.337_353delinsG and c.423+1G>A) were identified in the homologous alleles of the patients, respectively. Although these two variants were first identified in the Japanese population, they have since been reported in other Asian countries and might be founder mutations of the gene in Asian population.
•Aim of study was to determine UGT gene polymorphism and its impact on VPA and CBZ metabolism.•Homozygous and heterozygous variants of UGT genes were appreciably high in Khyber Pakhtunkhwa population ...of Pakistan compared to ethnic groups of other countries.•Genetic polymorphisms of UGT1A6 decreased the plasma levels of VPA.•Neither of the UGT1A6 and UGT2B7 polymorphisms revealed any significant shift in CBZ levels.
Ethnic variation is one of the important factors in clinical practice that may affect the pharmacokinetics of drugs. The present study aims to determine the distribution pattern of UGT1A6 and UGT2B7 gene polymorphism and its possible impact on the metabolism of valproic acid (VPA) and carbamazepine (CBZ) in patients with epilepsy from Khyber Pakhtunkhwa region of Pakistan. Methods: After the enrollment of targeted patients, blood was collected for genotype analysis through Sanger sequencing. Plasma concentrations of VPA and CBZ were determined by reversed-phase high-performance liquid chromatography (HPLC) at the follow-up visit of third month from the initiation of therapy. The drug plasma levels were correlated with different genotypes of UGT1A6 and UGT2B7 to determine the impact of genetic polymorphism on the drug metabolism. Results: Of the total 178 epileptic patients, 120 subjects were prescribed VPA monotherapy while 58 subjects were given CBZ monotherapy. The mean age of the subjects was recorded as 26.1 ± 13.5 years with a predominance of the male gender. Generalized tonic-clonic (GTC) was the most prevalent type of seizure (82%) followed by partial seizure. Genotype analysis revealed that the frequency of homozygous and heterozygous variants of the targeted UGT genes were exceptionally high in the Khyber Pakhtunkhwa population compared to the ethnic groups of other countries. In UGT1A6-A552C and UGT1A6-A541G, AC and AG were the most prevalent genotypes with respective frequencies of 43.2% and 41.1% whereas, in UGT2B7-T161C and UGT2B7-G211T, TC and GG were the most prevalent genotypes with respective frequencies of 42.7% and 99.4%. In the VPA-treated group, the homozygous and heterozygous variants of UGT1A6-A552C and UGT1A6-A541G were significantly associated with lower drug plasma concentrations (p < 0.05). However, none of the genotypes of UGT2B7-T161C revealed any significant association with VPA plasma concentration (p greater than 0.05). In the CBZ-treated group, UGT gene polymorphisms were not recognized to cause alteration in the drug plasma concentrations (p greater than 0.05). Conclusion: The genetic polymorphisms of UGT1A6, but not UGT2B7 significantly affected the plasma levels of valproic acid. The chosen SNPs did not reveal a role in determining the plasma levels of carbamazepine.
To summarize the clinical manifestations of craniofacial fibrous dysplasia (CFD) patients with ocular complications, and find effective methods to diagnose early.
Nine CFD patients with ocular ...complications, and their parents were recruited in this study. All patients underwent ocular and systemic examinations. Bone lesions from all patients and peripheral blood from patients and their parents were collected for whole exome sequencing (WES). According to the screening for low-frequency deleterious variants, and bioinformatics variants prediction software, possible disease-causing variants were found in multiple CFD patients. The variants were validated by Sanger sequencing. Trio analysis was performed to verify the genetic patterns of CFD.
All patients were diagnosed with CFD, according to the clinical manifestations, classic radiographic appearance, and pathological biopsy. The main symptoms of the 9 CFD patients, included visual decline (9/9), craniofacial deformity (3/9) and strabismus (2/9), with few extraocular manifestations. The family backgrounds of all the CFD patients indicated that only the patient was affected, and their immediate family members were normal.
variants were identified in all bone lesions from CFD patients, including two variant types: c.601C > T:p.R201C(6/9) and c.602G > A:p.R201H (3/9) in exon 8. The detection rate reached 100% by WES, but only 77.8% by Sanger sequencing. Interestingly, we found
variants could not be detected in peripheral blood samples from CFD patients or their parents, and other potentially disease-causing gene variants related to CFD were not found.
For CFD patients with bone lesions involving the optic canal or sphenoid sinus regions, ocular symptoms should also be considered. Furthermore, we confirmed that CFD is not inherited, somatic variants in the
gene are the main pathogenic gene causing CFD. Compared to the traditional methods in molecular genetic diagnosis of CFD, WES is more feasible and effective but limited in the type of samples.
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