Journal of Human Genetics and Genomics
Volume:3 Issue: 1, Jun 2019

Advances in nucleic acid based molecular diagnosis techniques, provided the basis for efficient, accurate and rapid detection of biological/pathological agents. However, these techniques suffer from major limitations including lack of the genomic sequence of high-risk pathogens to calibrate techniques as positive controls, false positive results and restrictions on access to existing commercial tests. However with the advent of synthetic biology, it is possible to construct appropriate positive controls. Artificial genetic constructs are the synthetic constructs that contain genetic materials from highly dangerous bacteria or viruses, which can be used as positive controls in molecular techniques such as PCR. In this review, we introduce positive controls and simulator positive controls firstly and discuss about artificial genetic constructs, procedure of design and synthesis secondly. Finally, we discussed about single and chimeric artificial genetic constructs as two main categories of these positive control.

In recent decades, infertility is becoming a public health issue. Male spermatogenesis failure has been considered a major contributory factor to infertility. Mammalian spermatogenesis is a well-defined process, requiring highly regulation processes in both transcriptional and the posttranscriptional levels. Discovery of microRNAs (miRNAs or miR) as essential class of gene expression regulators has provided new insights into a multitude of biological processes including spermatogenesis. In current review study, we first provide a short overview of spermatogenesis process, and then focus on recent studies that have elucidated the essential role of miRNAs in different steps of sperm production.

 Breast cancer is one of the most common types of cancers and the leading cause of death, especially in women throughout the world. Various genetics and environmental factors have been identified as a risk factor for development of breast cancer. Long non-coding RNAs (lncRNAs) have gained significant attention in recent years as new and crucial players in cancer development. Curcumin is a plant compound that has been shown to inhibit various aspects of cancer including apoptosis, inhibition of cell proliferation, invasion and angiogenesis.

 Elevated expression level of two lncRNAs, HULC and Linc-ROR, has been reported as contributing factor to the development of breast cancer. Therefore, we aimed to investigate the effect of nanocurcumin on expression levels of these genes. Besides, apoptotic effect of nanocurcumin on MCF7 cells was investigated.

 MCF7 cells were exposed to various concentrations of nanocurcumin and their metabolic activity was measured by MTT assay. The level of HULC and Linc-ROR genes was evaluated by real-time PCR. Apoptosis was also determined by Annexin test.

 Twenty micromollar (µm) concentrations of nanocurcumin significantly decreased the expression of HULC and Linc-ROR genes (P < 0.05). These concentrations also led to significantly higher apoptosis rate in MCF7 cells (97%) compared to cell treated with curcumin.

 Our findings suggested that nanocurcumin exhibits apoptotic effects on breast cancer MCF7 cells by reducing the expression level of two proto-oncogene lncRNAs, HULC and Linc-ROR, which are both involved in downregulation of p53.
 

 Burkholderia bacteria are species from protobacter and the pathogens of this species include Burkholderia mallei and pseudomallei causing glanders. The yellow fever virus is a blavovirus that has a single-stranded polyribonucleotide genome (positive strand). Yellow fever is a type of acute viral disease, also called black vomit. Even though considerable advances have been made in the development and use of molecular techniques for the detection of pathogens such as Burkholderia species and yellow fever, a major problem in these techniques is lack of proper positive control. Recently developed genetically modified vectors and simulator construct have provided engineered tools that can be used as a positive control in molecular diagnosis.

 In the current study, we first designed and produced chimeric simulator constructs, containing specific genes from Burkholderia mallei, pseudomallei, and yellow fever virus. Then, the operational efficiency of the designed structures was evaluated using a single and dual-mode quantitative (using SYBER Green) and qualitative (using the TaqMan probe) Real-time PCR.

 The minimum and maximum sensitivity of designed construct for Burkholderia and yellow fever detection was indicated to be 0.1 pg and 10 ng, respectively.

 Our findings suggest that this positive control constructs can be used for development of new diagnostic kits.
 

Chronic myeloproliferative disorders (CMPD) occur due toclonal proliferation of the single hematopoietic stem cells and result in an increased number of mature and immature cells in the peripheral blood. The mutations in JAK2 gene are identified in large numbers of CMPD patients.

The aim of this study was to investigate thep.V617F (c.1849G > T) mutation as well as exon 12 mutations in JAK2 gene in the CMPD patients.

Philadelphia chromosome negative CMPD patients were recruited for this study. In order to study p.V617F and JAK2 exon 12 mutations in JAK2 gene, FRET probe real-time PCR, allele specific PCR and PCR-direct sequencing were utilized.

JAK2 p.V617F mutation was found in polycythemia vera, Essential thrombocytosis and idiopathic myelofibrosis (67%, 52% and 50% respectively) but not in idiopathic erythrocytosis patients. Also no mutation was found in JAK2 exon 12 of these patients.

Our data regarding p.V617F was in concordance with the previous studies. The absence of any mutation in exon 12 of our patients may be due to extracting DNA from whole blood cells instead of granulocytes, that may impact the detection rate of cycle sequencing method.

 Beta thalassemia is an autosomal recessive genetic disease with the symptoms of severe anaemia, ineffective erythropoiesis and bone deformities. Preimplantation genetic diagnosis is a noninvasive clinical tool for couples who are at risk of affected pregnancy to have a healthy child.

 Here we report a PGD test for a couple who were heterozygous for CD36/37(-T) mutation in HBB gene and had terminated one affected pregnancy.

 Haplotype analysis of 6 flanking STR markers as well as variant detection by cycle sequencing were included in our PGD test in order to investigate the status of the embryos reliably.

 Three out of five embryos were transferable.

 One normal and one carrier embryo were transferred which resulted in the singleton pregnancy and the birth of a healthy girl.
 

Lowe syndrome is a condition that primarily affects eyes, brain, and kidneys. This disorder follows X-linked recessive mode of inheritance and it occurs in males mainly. Mutations in OCRL (located at Xq25) gene can cause accumulation of phosphatidylinositol bisphosphate and disturbed actin cytoskeleton remodeling. There are 268 mutations in OCRL gene causing Lowe syndrome or Dent disease 2 in HGMD database, however 10 - 20% of Lowe syndrome suspects remain undiagnosed at molecular level. Here we present a male case of Lowe syndrome with characteristic features. Comprehensive clinical examination and genetic counseling were performed. Sanger sequencing was employed to investigate the possible OCRL mutations and we identified a donor splice site variant (NM-000276: c.2469 + 1G > A) in hemizygous state. This is a pathogenic variant according to the ACMG standards and guidelines and might explain the clinical features of the patient. This result is in accordance with the clinical diagnosis of Lowe syndrome and it is absent from ExAC, 1000 G, Iranome, GME, gnomAD Genome databases of healthy controls. In-silico analysis of this splicing variant revealed that the position is highly conserved between species. Splicing prediction tools predicted some changes in splicing pattern of the OCRL transcript, elimination of some protein features, and malfunctioning the OCRL protein as a consequence of this variant. Accordingly, we proposed the c.2469 + 1G > A variant might explain the clinical features in studied patient and be employed for prenatal diagnosis of Lowe syndrome in the family.

 Steroid-resistant nephrotic syndrome is a genetic disease with autosomal recessive inheritance pattern and symptoms such as proteinuria and hypoalbuminemia and rapid progress of kidney disease. Preimplantation genetic diagnosis is an option for couples who are at risk of affected pregnancy to have a healthy child.

 This study aimed to develop a new PGD test for a couple who are heterozygous for a mutation in NPHS2 gene and have a son affected to steroid-resistant nephrotic syndrome.

 Variant detection by cycle sequencing and Multiplex fluorescent PCR for identification of flanking STR markers were used to investigate the status of the embryos.

 Three out of six embryos were transferable from which one was transferred and resulted in the birth of a healthy boy.

 We recommend increasing the number of the STR markers to two at the downstream of the NPHS2 gene especially in cases that direct mutation analysis such as cycle sequencing is not applied.