Biochemical Genetics Research Articles

Urinary pyridinoline cross-links as biomarkers of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a group of genetic heterogeneous connective tissue disorders characterized by increased bone fragility and susceptibility to fractures. Laboratory diagnosis relies on time-consuming and cost-intensive biochemical and molecular genetics analyses. Therefore, it is desirable to identify and establish new diagnostic markers for OI that are reliable, cost-effective and easily accessible. In our study we have identified the ratio of the urinary pyridinoline cross-links lysyl-pyridinoline and hydroxylysyl-pyridinoline as a promising, time- and cost-effective biomarker for osteogenesis imperfecta, that could be used furthermore to investigate cases of suspected non-accidental injury in infants.

Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication.

We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5's conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication.

Model super-organisms: Can the biochemical genetics of E. coli help us understand aging?

Escherichia coli is a powerful model organism to help us understand biochemical pathways and enzyme function. In E. coli, biosynthetic pathway mutants are conditionally viable depending on the composition of the nutrient media. Genetic analysis allows enzyme function to be linked to gene sequences. E. coli is used in the lab as a food source for the nematode worm Caenorhabditis elegans, a model organism for the understanding of basic animal biology. Recent work has shown that specific biosynthetic pathways in E. coli can influence C. elegans aging. Careful experimentation is needed to determine whether E. coli biochemistry influences aging by altering C. elegans nutrition or through changes to bacterial functions, such as toxin production. Understanding these interactions in the C. elegans–E. coli model “super-organism” will inform studies of how bacteria of the human microbiota interact with the host.

Annual Report on the External Quality Assessment Scheme for Biochemical Genetics in Korea (2014)

Annual Report on the External Quality Assessment Scheme for Biochemical Genetics in Korea (2014)

Biomarkers for disease progression of primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown origin. There is no medical treatment of proven benefit on survival; once patients have progressed to end-stage liver disease, the only treatment option is liver transplantation. Over the last years, some progress has been made in identifying biomarkers of PSC disease progression. Categories that can be distinguished include clinical and biochemical biomarkers, histology, imaging, prognostic modelling and genetics. With this review, we summarize biomarkers for progression of PSC from these six categories, which have been studied to date. Biomarkers for the progression of PSC disease course can be used for several purposes. First of all, they can be implemented as surrogate endpoints for clinical trials. Second, biomarkers of disease progression form the basis of prognostic modelling, which is needed for proper patient counselling and management. Lastly, these biomarkers may yield a better understanding of PSC pathogenesis.

Squalestatin is an inhibitor of carotenoid biosynthesis in Plasmodium falciparum.

The increasing resistance of malaria parasites to almost all available drugs calls for the characterization of novel targets and the identification of new compounds. Carotenoids are polyisoprenoids from plants, algae, and some bacteria, and they are biosynthesized by Plasmodium falciparum but not by mammalian cells. Biochemical and reverse genetics approaches were applied to demonstrate that phytoene synthase (PSY) is a key enzyme for carotenoid biosynthesis in P. falciparum and is essential for intraerythrocytic growth. The known PSY inhibitor squalestatin reduces biosynthesis of phytoene and kills parasites during the intraerythrocytic cycle. PSY-overexpressing parasites showed increased biosynthesis of phytoene and its derived product phytofluene and presented a squalestatin-resistant phenotype, suggesting that this enzyme is the primary target of action of this drug in the parasite.

El laboratorio en el diagnóstico de las enfermedades metabólicas hereditarias. Impacto de las nuevas tecnologías

Inherited metabolic diseases are a group of more than 600 diseases classified according to the altered metabolic pathway and their pathogenesis. Most are diagnosed postnatally after recognition by a number of clinical symptoms suggestive of disease. Laboratories of biochemical and molecular genetics are involved in the recognition of these diseases as the analysis of metabolites, proteins and genes are key for diagnosis. This article reviews how (approach) these diseases are diagnosed and the biochemical and genetic laboratory techniques commonly used. It is in the area of identification of metabolites (mass spectrometry) and mutation detection (massive sequencing) where the impact of new technology in recent years has been spectacular, which facilitated rapid diagnosis with minimally invasive tests and will facilitate future population screening.

The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.

Expression of B-cell activating factor in acute lymphoblastic leukemia patients

Introduction Acute lymphoblastic leukemia (ALL) is a biologically heterogeneous disorder needing determination of morphologic, immunologic, cytogenetic, biochemical, and molecular genetics characterizations of lymphoblasts to establish diagnosis. Microenvironmental cues play critical roles in cancer biology and malignant cells are responsive to multiple extrinsic factors. There is emerging evidence that B-cell activating factor (BAFF) is a critical factor for the growth and survival of both normal and malignant clones of B-cells and can augment tumor cell growth. There is evidence that B-lineage neoplasms have aberrant expression of BAFF. Objectives The aim of this study was to evaluate serum BAFF levels to assess its prognostic impact on ALL and correlate it with mortality. Patients and methods The study was conducted at Ain Shams University on 40 ALL pediatric and adult patients, in addition to 20 controls. Patients underwent detailed history and clinical examination, bone marrow examination, and evaluation of serum BAFF levels using enzyme-linked immunosorbent assay. Thereafter, they were assessed for treatment response and patients' mortality on day 28 from induction of chemotherapy and after 6 months, respectively. Results We found a statistically significant lower BAFF levels among controls compared with the patient group, in which there was no significant difference between pediatric and adult subgroups. High serum BAFF was significantly correlated to patients' poor treatment response but not mortality. Conclusion High BAFF in our studied patients may be explained by its role in augmenting B-cell tumor growth. In our study, we found that BAFF had no relation with disease outcome. This may be caused by the limited number of patients included. Assessment of BAFF level at the time of diagnosis may be a predictor for response to treatment, as we found a significant relation between BAFF level and response to treatment. This finding recommends that patients with high BAFF level at the time of diagnosis be subjected to intensified course of therapy.

Scandinavian mutation research in barley - a historical review.

In 1928, the Swedish geneticists Hermann Nilsson-Ehle and Åke Gustafsson started on their suggestion experiments with induced mutations using the barley crop. In 1953, at the instigation of the Swedish Government, the 'Group for Theoretical and Applied Mutation Research' was established. Its aim was to study basic research problems in order to influence and improve methods for breeding cultivated plants. The research was non-commercial, even if some mutants were of practical importance. The peaks of activities occurred during the 1950s, 1960s and 1970s. Applying X-rays and UV-irradiation very soon the first chlorophyll mutations were obtained followed by the first viable mutations 'Erectoides'. Soon the X-ray experiments expanded with other types of irradiation such as neutrons etc. and finally with chemical mutagens, starting with mustard gas and concluding with the sodium azide. The research brought a wealth of observations of general biological importance, high increased mutation frequencies, difference in the mutation spectrum and to direct mutagenesis for specific genes. A rather large collection of morphological and physiological mutations, about 12 000 different mutant alleles, with a very broad variation were collected and incorporated into the Nordic Genetic Resource Center (NordGen) Sweden. Barley, the main experimental crop has become one of the few higher plants in which biochemical genetics and molecular biological studies are now feasible. The collection is an outstanding material for mapping genes and investigating the barley genome. Several characters have been studied and analyzed in more detail and are presented in this historical review.

Modern diagnostic approach to hereditary xanthinuria.

Hereditary xanthinuria (HX) is a rare inherited disorder caused by a deficiency of xanthine dehydrogenase/oxidase (XDH/XO). Missing XDH/XO activity leads to undetectable levels of uric acid excessively replaced by xanthine in serum/urine. The allopurinol loading test has been traditionally used to differentiate between HX types I and II. Final confirmation of HX has been based on the biopsy finding of the absent XDH/XO activity in the small intestine or liver. We present the clinical, biochemical, ultrasound and molecular genetics findings in three new patients with HX and suggest a simple three-step approach to be used for diagnosis, typing and confirmation of HX. In the first step, the diagnosis of HX is determined by extremely low serum/urinary uric acid excessively replaced by xanthine. Second, HX is typed using urinary metabolomics. Finally, the results are confirmed by molecular genetics. We advocate for this safe and non-invasive diagnostic algorithm instead of the traditional allopurinol loading test and intestinal or liver biopsy used in the past.

Early identification of treatable inborn errors of metabolism in children with intellectual disability: The Treatable Intellectual Disability Endeavor protocol in British Columbia.

Intellectual disability (ID) is a lifelong, debilitating condition affecting 2% to 3% Canadians (1). It is defined by deficits in cognitive functioning (IQ 2 developmental domains, eg, fine/gross motor skills, speech and interaction) (2,3). In Canada, approximately 7600 to 11,500 children are born with GDD annually (1). In the present commentary, the term ID will be used to refer to ID/GDD.

Fifteen years experience: Egyptian metabolic lab

BackgroundInborn errors of metabolism (IEM) are single gene disorders responsible for abnormalities in the synthesis or catabolism of proteins, carbohydrates and fats by means of defective enzymes or transport proteins which results in a block of the metabolic pathway and accumulation of metabolites in different tissues. This study shows the most common diagnosed inherited inborn errors of metabolism among the Egyptian population. Prior to 1995, the diagnosis of inherited metabolic disorders in Egypt was very limited and diagnosed mainly on clinical suspicion. In 1995, The Biochemical Genetics Unit at The National Research Centre has been established as a part of The Human Genetics Department and later on in 2003 it was developed into The Biochemical Genetics Department by applying advanced techniques and equipments and providing early diagnosis for the metabolic disorders which led to better outcome in our patients. Material and methodsWe have retrospectively reviewed a total of 12,148 cases suspected to have inborn errors of metabolism (IEM) with different age groups. They had been referred from several diagnostic centers and hospitals in Egypt to The Department of Biochemical Genetics at The National Research Centre. The diagnosis of these disorders was confirmed by qualitative determination of amino acid profile, quantitative determination of phenylalanine and galactose levels using dried blood spots (DBSs), quantitative determination of urinary glycosaminoglycans (GAGs), two-dimensional electrophoretic separation of GAGs in urine and the assay for lysosomal enzymes activities in plasma and leukocytes. ResultsOut of the total number of cases; 1041 (8.6%) patients were proved to have metabolic disorders. Those patients were classified as: 722 patients (69.4%) with lysosomal storage disorders, 302 patients (29%) with amino acid disorders and 17 patients (1.6%) with galactosemia. ConclusionThis study illustrates the experience of the reference metabolic lab in Egypt over 15years. The lab began metabolic disorder screening by using simple diagnostic techniques like thin layer chromatography and colored tests in urine which by time updated and upgraded the methods to diagnose a wide range of disorders. This study shows the most common diagnosed inherited inborn errors of metabolism among the Egyptian population.

Molecular genetic testing for cystic fibrosis: laboratory performance on the College of American Pathologists external proficiency surveys.

Molecular testing for cystic fibrosis mutations is widespread and routine in reproductive decision making and diagnosis. Our objective was to assess the level of performance of laboratories for this test. The College of American Pathologists administers external proficiency testing with multiple DNA samples distributed biannually. RESULTS are analyzed, reviewed, and graded by the joint College of American Pathologists/American College of Medical Genetics and Genomics Biochemical and Molecular Genetics Committee. Assessment is based on genotype and associated clinical interpretation. Overall, 357 clinical laboratories participated in the proficiency testing survey between 2003 and 2013 (322 in the United States and 35 international). In 2013, US participants reported performing nearly 120,000 tests monthly. Analytical sensitivity and specificity of US laboratories were 98.8% (95% confidence interval: 98.4-99.1%) and 99.6% (95% confidence interval: 99.4-99.7%), respectively. Analytical sensitivity improved between 2003 and 2008 (from 97.9 to 99.3%; P = 0.007) and remained steady thereafter. Clinical interpretation matched the intended response for 98.8, 86.0, and 91.0% of challenges with no, one, or two mutations, respectively. International laboratories performed similarly. Laboratory testing for cystic fibrosis in the United States has improved since 2003, and these data demonstrate a high level of quality. Neither the number of samples tested nor test methodology affected performance.

Annual Report on External Quality Assessment of Biochemical Genetics in Korea (2013)

Annual Report on External Quality Assessment of Biochemical Genetics in Korea (2013)

Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster

The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster.

The Golgi Arf‐GEFs Gea1 and Gea2 are regulated by their HDS3 domains and the Rab GTPase Ypt1 (LB157)

The Golgi Arf GTPases control the sorting of membranes and proteins through all eukaryotic cells, recruiting adapters and cargo to sites of vesicle formation when in their activated GTP‐bound form. Arf guanine nucleotide exchange factors (Arf‐GEFs) are the master regulators of Arf activation and are conserved throughout eukaryotes. Two Arf‐GEFs, Gea1 and Gea2, function at the cis‐Golgi in Saccharomyces cerevisiae, but we know relatively little about how these GEFs are regulated. Recent work with their close relative at the trans‐Golgi network, Sec7, has revealed that recruitment of Sec7 to the trans‐Golgi network and relief of its autoinhibition are dependent on its C‐terminal domains, as well as on positive feedback from Arf1 and other protein interactions. The Golgi Arf‐GEFs share predicted domain architecture, suggesting that Gea1/2 may be regulated by mechanisms similar to those of Sec7. However, the specific proteins which orchestrate the activation and localization of Sec7 do not appear to act upon Gea1/2. In vitro biochemical studies, genetics, and live cell microscopy have revealed two contributing factors in Gea1/2 regulation: an interaction with the Rab GTPase Ypt1 and the regulatory roles of one of the domains of Gea1/2. Combined with structural studies, these results will define general features of Golgi Arf‐GEF regulation and localization, as well as features specific to Gea1 and Gea2, providing fundamental insight into how Arf1 is regulated at the Golgi.Grant Funding Source: Supported by NIH/NIGMS grant R01GM098621 and NIH grant T32GM007273

Evolution of a hot genome

The chile pepper has a long and important human history. Chiles, members of the plant genus Capsicum in the Solanaceae family, evolved in the New World and were domesticated there several millennia ago (1). Although it is hard to imagine a Mexican, Szechuan, or Indian dinner without chiles, these characteristic cuisine elements are a relatively recent development, having only achieved their enormous Old World culinary impact in the centuries after Columbus. Indeed, several other Solanaceae crops are of entirely New World origin, with tomato ( Solanum lycopersicum ) and potato ( Solanum tuberosum ) of particular importance, but also pepino ( Solanum muricatum ) and tomatillo ( Physalis philadelphica ) (Fig. 1 A ). In testimony to the importance of this plant group, whole-genome sequences of tomato (2) and potato (3) were among the first 50 plant genomes available for study. Now, we can add chile pepper to the mix, as published in PNAS by Qin et al. (4) and independently by Kim et al. (5). Although both papers provide excellent resources to the plant science community and share several similar findings (e.g., on the enormous expansion of the chile pepper genome by proliferation of mobile elements and on the biochemical genetics of the fruit’s characteristic pungency), the work of Qin et al. (4), which includes considerable genomic resequencing of multiple cultivated varieties and a wild pepper, the chiltepin, provides important added data for evaluating the (in part artificially derived) adaptive landscape of cultivated peppers. Fig. 1. The genome of chile pepper in an evolutionary context. ( A ) A phylogeny of lamiid angiosperms, the family Solanaceae in yellow and chile pepper in red. ( B ) Syntenic dotplot of tomato chromosome 2 (vertical) versus chiltepin pepper chromosomes 2 ( Upper ) and 4 ( Lower ), with syntenic gene duplicates colored according to their Ks values (synonymous substitutions per synonymous site), as plotted in the x axis of C … [↵][1]1To whom correspondence should be addressed. E-mail: vaalbert{at}buffalo.edu. [1]: #xref-corresp-1-1

Investigation on mechanism of sterility of male hybrids between yak and cattle

Cattle–yak, the first filial generation of yak and ordinary cattle, showed obvious hybrid vigor, but a difficult problem in crossbreeding and improvement of yak is that the males are sterile. Thus it is not possible to steadily utilize the heterosis. So the study of the mechanism of male sterility is still one of the most interesting fields in yak science. Therefore, this paper will review contemporary studies in cattle–yak male sterility including the process of spermatogenesis, development of genital organs, pituitary and endocrinology, biochemical genetics and candidate genes correlating spermatogenesis, etc.

Next Generation DNA Sequencing and Its Application in Clinical Medicine

the reprints of 20 articles were obtained to be used for the references for this review paper. In the beginning of the 20th century, Mendel’s law of inheritance could explain the recurrence of certain diseases in families and it was recognized by Sir Garrod et al. From this point and on, medical genetics grew from a tiny subspecialty dealt with rare hereditary diseases to a fully recognized medical specialty. Medical genetics’ concept and approaches are important components of the diagnosis and management of nearly all disorders. Medical genetics was a front line specialty which believed in evidence based medicine. The diagno sis of genetic disorders is usually proved or supported by genetic testing. Hemoglobin electro phoresis, lipid profiles, karyotype and gene se quences are examples. Biochemical genetics, cytogenetics, and mole­ cular genetics are the 3 recognized laboratory subspecialties by American College of Medical The editor in chief of the Korean Journal of Peri­ natology, Hee­Sup Kim, M.D., Ph.D., invited me to write a review article titled “Next Genera tion DNA Sequencing and Its Application in Clinical Medi­ cine”. In order to write clinically useful re view paper for actively practicing physicians in perinatal medicine area, Medline search under the keyword of next generation DNA sequencing was confined to Journal of the American Medical Association, New England Journal of Medicine, Obstetrics and Gyne­ cology, Pediatrics, Journal of Pediatrics, Journal of Obstetrics and Gynecology, Obstetrics and Gyne­ cology, Genetics in Medicine, and Journal of Peri­ natology which were published between January 01, 2004 and June 30, 2014. Upon careful review 대 한 주 산 회 지 제25권 제3호, 2014 Korean J Perinatol Vol.25, No.3, Sep, 2014 http://dx.doi.org/10.14734/kjp.2014.25.3.133