Evaluation and Application of Screening Strategies for Point Mutations in the Retinoblastoma Gene

The present study aimed to evaluate two screening strategies, single strand conformation polymorphism (SSCP) and amplification mismatch detection (AMD) analysis, for the detection of point mutations in the retinoblastoma gene (RB). SSCP was optimised and applied to exons 12-22 of the RB gene which constitute the most important functional domain. Leukocyte DNA from 20 patients with bilateral retinoblastoma (Rb), tumour DNA from 40 patients with bladder carcinoma and tumour DNA from 39 patients with breast carcinoma were subjected to SSCP analysis. SSCP band shifts were found in 4 of 20, 1 of 40 and none of 39 patients respectively. AMD was optimised and applied to exons 12-16 of the RB gene and also to reverse-transcriptase PCR in the 20 patients with bilateral Rb. Cleavage was found in 2 patients; one was found in a cDNA segment and the other was found in genomic DNA. Neither of these patients corresponded to the 4 with SSCP band shifts. Thus in total, 6 patients with Rb and one with bladder carcinoma had mutations detected and proof was sought by sequencing. Amplification of segment C of the cDNA of patients with bilateral Rb has revealed that patient EAS showed an additional band indicating either a deletion or a splice mutation. Analysis of exon 17 and the flanking intron of the same patient with AMD showed a cleavage with hydroxylamine. Sequencing of the exon revealed that the mutation is a C substitution of the A at position -2 of the acceptor site of intron 16, impairing normal splicing of the RNA. The mutation results in skipping of exon 17, because the short transcript band on the agarose gel was approximately 196 bp shorter than the original band and exon 17 was 196 bp in size. This leads in turn to the production of a truncated RB protein. By analogy to other published mutations, this aberrant, destabilised protein might not be able to bind the E1A oncoprotein. In addition, the mutant RB protein may fail to complex with SV40 large T antigen. Analysis of segment C of the cDNA from patient PC with bilateral Rb showed a cleavage with hydroxylamine reaction. Sequencing of the segment revealed the mutation to be a T→G transversion at nucleotide position 1587 within exon 16 causing a substitution of histidine to glycine. The missense mutation may or may not have a functional effect. However, this residue lies within an RB domain (aminoacids 393-572) identified recently by in vitro deletion mutants to be required for oncoprotein binding. This mutation creates a restriction site for Nde I. Sequencing of exon 21 from patient MH who had an SSCP band shift, revealed that an insertion of a G at nucleotide position 2251 within exon 21 resulted in a novel stop codon (TAA) at codon 719 (nucleotide position 2295) within exon 21 thus deleting the domain interacting with the SV40 T antigen. The translated protein is most probably too short to be functional. To confirm whether the observed SSCP pattern in the region of exon 21 of the RB gene was a new germ line mutation or inherited from one of the parents, heteroduplex analysis of the parents revealed either the mutation was de novo or one of the parents had germ line mosaicism. In addition, the change creates a restriction site for the restriction enzyme FokI. Another mutation detected (from patient AR) by SSCP analysis was an A to C transversion at position 1636 in exon 16 causing AGA to CGA codon change, both coding for the same amino acid: Arginine, this mutation abolishes a restriction site for the restriction enzyme CvijI. The mutation site was located in the last base of the exon 16, although it has not been shown in this study in mRNA, it could affect splicing of the mRNA of RB gene. Two mutations found were considered to be silent mutations, because they do not cause any amino acid change. Their RNA transcripts were found to be normal. These mutations were a T to C transition at position 1617 in exon 16 (Patient GM) which alters the codon GTT (GUU) to GTC (GUC) both coding for the same amino acid; Valine and an A→G transition in intron 19 (patient EAS) which abolishes a restriction site for the restriction enzyme Tsp 509 I which may be useful in tracking this mutation in affected family members. Analysis of 100 samples (patients with bilateral Rb and other tumours) for this restriction site revealed that none of them has same change. The mutation found from the patient with bladder carcinoma was a G to C transversion at position +1 of the donor site of intron 12, probably impairing normal splicing of the RNA. The second mutation was assumed to lie in a part of the retinoblastoma gene that was not analysed, since in somatic cases two hits in the RB gene are expected. Intact RNA could not always be recovered from the clinical material used in this study, therefore the diagnostic strategy for bladder carcinoma was chosen not to be based on the analysis of RNA transcript. The change creates a recognition site for the restriction enzymes MaeII, Bpu101, DdeI. The seven mutations detected in this study were all novel and emphasise the heterogeneity of the molecular pathology in this gene

Advances in biotechnology: genomics and genome editing

Genomics, the study of genes, their functions and related techniques has become a crucial science for developing understanding of life processes and how they evolve. Since the advent of the human genome project, huge strides have been made in developing understanding of DNA and RNA sequence information and how it can be put to good use in the biotechnology sector. Newly derived sequencing and bioinformatics tools have added to the torrent of new insights gained, so that 'sequence once and query often' type DNA apps are now becoming reality. Genome editing, using tools such as CRISPR/Cas9 nuclease or Cpf1 nuclease, provide rapid methods for inserting, deleting or modifying DNA sequences in highly precise ways, in virtually any animal, plant or microbial system. Recent international discussions have considered human germline gene editing, amongst other aspects of this technology. Whether or not gene edited plants will be considered as genetically modified remains an important question. This will determine the regulatory processes adopted by different groups of nations and applicability to feeding the world's ever growing population. Questions surrounding the intellectual property rights associated with gene editing must also be resolved. Mitochondrial replacement therapy leading to '3-Parent Babies' has been successfully carried out in Mexico, by an international team, to correct mother to child mitochondrial disease transmission. The UK has become the first country to legally allow 'cautious use' of mitochondrial donation in treatment. Genomics and genome editing will continue to advance what can be achieved technically, whilst society determines whether or not what can be done should be applied

Prediction, prevention and personalisation of medication for the prenatal period: genetic prenatal tests for both rare and common diseases

Genetic testing usually helps physicians to determine possible genetic diseases in unborn babies, genetic disorders of patients and the carriers who might pass the mutant gene on to their children. They are performed on blood, tissues or other body fluids. In recent years, the screening tests and diagnostic tests have improved quickly and, as a result, the risks of pregnancy can be determined more commonly and physicians can diagnose several genetic disorders in the prenatal period. Detecting the abnormalities in utero enables correct management of the pregnancy, prenatal and postnatal medical care, and it is also important for making well informed decisions about continuing or terminating a pregnancy. Besides the improvements of conventional invasive diagnostic tests, the discovery of circulating cell-free foetal nucleic acids in maternal plasma has developed a new point of view for non-invasive prenatal diagnosis recently

О параметрах системы подготовки принятия решений государственной организации с помощью бизнес-процессов

В статье приводится описание параметров, необходимых для информационной системы принятия решений государственной организации при оказании услуг с помощью бизнес-процессов. Любая информационная система, позволяющая подготавливать данные для принятия решений, строится на основе количественной информации. Однако, само решение выбирается чаще всего на основе опыта, знаний, что субъективно и не во всех случаях является правильным. Нами предлагается выделить класс событий, для которых возможно разработать шаблоны решений. Выбор решения основывается на анализе параметров бизнес-процессов государственной организации при оказании услуг.The article describes the parameters necessary for the decision-making information system of the state organization in the provision of services through business processes. Any information system that allows data to be prepared for decision-making is based on quantitative information. However, the decision itself is chosen most often on the basis of experience, knowledge, which is subjective and not always correct. We propose to allocate a class of events for which it is possible to develop decision templates. The choice of the solution is based on the analysis of the parameters of the business processes of the state organization in the provision of service

Periconceptional Mediterranean Diet during Pregnancy on Children’s Health

During pregnancy, rapid and subtle physiological changes are observed from conception to birth. Nutrition and other lifestyle factors before and during pregnancy have been shown in the literature to influence the health of both mother and child. A healthy and varied diet during pregnancy can provide adequate energy and nutrients for both the mother and the growing fetus. Current research focuses on the periconceptional phase, which includes the early processes of gametogenesis, embryogenesis and placentation. A variety of abnormalities and pregnancy-related problems occur during this period, including congenital defects, fetal loss, miscarriage and preterm birth. A varied and balanced diet during periconception is important to maintain fetal development and growth. To date, numerous studies have been conducted to investigate the effects of consuming different nutrients, foods or food groups during pregnancy on the health of mother and child. For example, the Mediterranean diet is considered as a balanced, nutrient-rich diet due to the low consumption of meat products and fatty foods and the high consumption of vegetables, cheese, olive oil, fish, shellfish and little meat. While many studies have been conducted in the literature to investigate the effects of a Mediterranean diet during pregnancy on fetal health, the results have been inconclusive. The aim of this article is to review the current literature on the Mediterranean diet during pregnancy

Genetic testing for tetralogy of Fallot

Abstract Tetralogy of Fallot (ToF) combines congenital cardiac defects including ventricular septal defect, pulmonary stenosis, an overriding aorta and right ventricular hypertrophy. Clinical manifestation of this defect depends on the direction and volume of shunting of blood through the ventricular septal defect and the associated right ventricular and pulmonary artery pressures. ToF accounts for 3-5% of congenital heart defects or 0.28 cases every 1000 live births. ToF has autosomal dominant inheritance. This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials

Genetic testing for Ebstein anomaly

Abstract Ebstein anomaly (EA) is a rare congenital tricuspid valve malformation, characterized by downward displacement of the septal leaflet and an atrialized right ventricle. About 80% of cases of EA are non-syndromic; in the other 20%, the anomaly is associated with a chromosomal or Mendelian syndrome. The prevalence of EA is estimated at about 1 per 20,000 live births, and accounts for less than 1% of all congenital heart defects. EA has autosomal dominant inheritance. Likely causative genes are: NKX2-5, MYH7 and TPM1. This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, potential risk assessment and access to clinical trials

Genetic testing for coarctation of aorta

Abstract Coarctation of the aorta (CoA) is an inherited narrowing of the proximal descending thoracic aorta. Histological features include localized medial thickening and infolding with superimposed neointimal tissue. CoA is diagnosed by detection of a murmur or hypertension during routine examination. Typical clinical features are delayed or absent femoral pulses and difference in blood pressure between the arm and legs. These symptoms may appear in the first weeks of life or after the neonatal period. CoA accounts for 4-6% of all congenital heart defects and has a reported prevalence of about 4 per 10,000 live births. It is more common in males than females (59% vs 41%). This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials

Genetic testing for ventricular septal defect

Abstract Ventricular septal defects (VSDs) are the commonest heart malformations and may affect the membranous or the muscular septum. Clinical presentation depends on the amount of interventricular flow, which is determined by the size of the defect and the relative resistances of the pulmonary and systemic vascular beds. The prevalence of VSD is estimated at about 5% among infants. Many small malformations present at birth may later undergo spontaneous closure. VSD may have autosomal dominant or autosomal recessive inheritance and may exist as isolated forms or as part of a syndrome. This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials