Genetics Laboratory Research Articles

Quantity and Quality Test of DNA Marigold Plants (Tagetes erecta L.) for the Suistainability of Plant Breeding

Marigold is one of the potential commercial flowers and demand continues toincrease. The high demand for marigold plants among the community so that the need for mutation plant breeding for marigold plants. It is hoped that giving mutations, it can increase genetic diversity in marigold plants which will be used as material for plant breeding activities to form new superior marigold varieties. This study aimed to examine the quantity and quality of mutant DNA marigold plants for the suistainabiliy of plant breeding. This research was conducted in the molecular genetics laboratory, Faculty of Agriculture, Universitas Sumatera Utara. The DNA of 5 mutant samples of marigold plants was isolated using the CTAB extraction method. The results showed that the lines that had bright and thick band patterns were found in samples P0, P2, P3 and P4 while thin and less bright bands were found insample P1. The purity of the resulting DNA ranged from 1.84 to 2.00 and the concentration of the resulting DNA ranged from 30.2 to 53.7 g/ml.

Results of the comprehensive study in newborn babies with congenital phenylketonuria

The timely detection of newborns with congenital phenylketonuria, in which the metabolism of the amino acid phenylalanine is disturbed due to the lack of the enzyme phenylalanine hydroxylase, remains an urgent issue. Increase in the level of phenylalanine and its toxic products in the cell leads to severe brain damage, which manifests itself in the form of mental retardation. Prompt diagnosis of phenylketonuria can prevent severe dementia and serious mental disorders. The aim of the work is a comprehensive study of newborns with congenital phenylketonuria for which enzymatic immunoassay, real-time PCR, tandem mass spectrometry were applied. Out of 5,293 newborns screened at the genetic laboratory of LPP “Center for Molecular Medicine” (Almaty, Kazakhstan) in 2019, two newborns were diagnosed with congenital phenylketonuria. The molecular genetic study in both of them indicated the presence of the R408W mutation in the heterozygous state in phenylalanine hydroxylase PAH gene. Upon application of the appropriate dietary therapy, the concentrations of phenylalanine in the blood reached 62.9 µM/L in the 1st and 173 µM/L in the 2nd newborn, which corresponds to its reference value. For the effective treatment of congenital phenylketonuria with a confirmed diagnosis based on molecular genetic studies (detection of a mutation in the PAH gene) in newborns, it is proposed to conduct additional biochemical studies for possible metabolic disorders.

Biotin-responsive Multiple Carboxylase Deficiency (MCD).

This study aimed to determine the clinical spectrum and biochemical findings on urine organic acids (UOA) in Biotin-responsive multiple carboxylase deficiency (MCD) patients presenting to the biochemical genetics laboratory (BGL). Patients reported as MCD, from January 2013-December 2020 were included. The UOA was analysed by gas chromatography mass spectrometer. Demographic, clinical, and biochemical details were extracted from the BGL history form. Two hundred and two patients were reported to have biotin responsive MCD with 111(55%) males and a median (Q3-Q1) age of 7 months (13-4). Of these 71.7% (n=145) patients presented in infantile period. Parental consanguinity was observed in 80% (n=161) and another 32.6% (n=66) cases grandparents were cousins. The main presenting features were seizures, developmental delay, and lethargy. The common peaks were determined on UOA 3OHIVA, MC and MCC. MCD is not rare in Pakistani population; it is recommended to include this disorder in newborn screening programs. Key Words: Biotin responsive multiple carboxylase deficiency, Organic acids, Amino acids, Pakistan, Inborn errors of metabolism.

Ethical, legal, and sociocultural challenges of genomic research in Jordan: a mixed methods study in patients with breast cancer with Jordanian–Palestinian heritage

BackgroundBreast cancer is a leading cause of morbidity and mortality in women worldwide. At King Hussein Cancer Center (KHCC), Amman, Jordan, 38% of breast cancer diagnoses are in women younger than 45 years, which might reflect a high prevalence of hereditary breast cancer. The aim of this study was to assess the contribution of germline mutations in BRCA1 and BRCA2 to breast cancer treated at KHCC. We also describe our experience in dealing with this delicate cultural issue in a tribal-based country with Palestinian–Jordanian family origins. MethodsPatients treated at KHCC with stage I–IV breast cancer, with a high-risk profile according to US National Comprehensive Cancer Network guidelines, were invited to participate. Patients were interviewed for consent and had full autonomy to decide whether they wanted to know their test result, inform their treating physician, or have a copy of the result placed in their charts. Charts were reviewed for clinical data and tumour pathology. A 10 mL blood sample was obtained for DNA extraction. A detailed three-generation family history was obtained. BRCA sequencing was done at the Myriad Genetics laboratory using BRCAnalysis. BRCA mutations were classified as deleterious, suspected deleterious, variant of uncertain significance (VUS), or favour polymorphism (harmless). The report received for each mutation result contained a detailed description of level of risk associated with the involved variant and estimated lifetime risk of breast and ovarian cancer, risk of second breast cancer or subsequent ovarian cancer, and risk of cancer to first, second, and third-degree family members. Patients were invited to an ad-hoc clinic with the treating physician for disclosure of results and counselling about estimated cancer risk to patient and relatives. Interviews with patients were summarised as text and qualitiative analysis for common themes was done. Challenges encountered during patient consent and disclosure of results were documented, as well as proposed solutions. Ethical approval was granted by the KHCC Institutional Review Board (no 11 KHCC 63). FindingsThe first 102 patients to meet inclusion criteria were invited to participate in the study, of whom 100 enrolled and provided written informed consent. Two patients declined to participate (a response rate of 98%). Six participants were residents of the West Bank and treated at KHCC, and 66 were from families of Palestinian origin residing in Jordan. The remaining patients were Jordanians. Median age was 40 years (range 22–75). 20 patients had deleterious mutations and seven had suspected deleterious mutations in BRCA1 and BRCA2 genes. Seven VUS were detected. All patients with BRCA1 and BRCA2 mutations had a significant family history of breast or ovarian cancer. Nevertheless, many patients with a significant family history (62 of 100) tested negative for BRCA1 and BRCA2 mutations. Many ethical dilemmas were highlighted during patient encounters, mainly relating to the guarantee of confidentiality and privacy in a tribal-based culture, fears of false reassurance, autonomy versus beneficence, and the bounds of physician–patient confidentiality when relatives are at genetic risk of cancer. Potential insurance, employment, and social discrimination implications were also addressed with the patients before testing and in more detail after disclosure of positive mutation results, which flagged a legal void in local genetic testing and data protection regulations. InterpretationOur cohort showed an important incidence of deleterious and suspected deleterious BRCA1 and BRCA2 mutations, suggesting that genetic testing should be discussed and offered to patients with high-risk features. Many high-risk patients tested negative for BRCA1 and BRCA2 mutations, therefore future studies should aim to test for mutations in other breast cancer susceptibility genes (eg, CHEK2, PALB2, and BRIP1). Running culturally sensitive genetic research in a country with limited resources is a challenging exercise and highlights the urgent need for guiding regulations. We recommend establishing a clinical cancer genetics programme, through which unaffected family members would benefit from early breast cancer screening and appropriate risk-reduction measures. FundingSister Institution Network Fund of MD Anderson Cancer Center and KHCC.

The Human Genome Project as a Singular Episode in the History of Genomics

In this paper, we progressively de-center the Human Genome Project (HGP) in the history of genomics and human genomics. We show that the HGP, understood as an international effort to make the human reference genome sequence publicly available, constitutes a specific model of genomics: prominent and influential but nevertheless distinct from others that preceded, existed alongside, and succeeded it. Our analysis of a comprehensive corpus of publications describing human DNA sequences submitted to public databases from 1985 to 2005 reveals a plethora of authoring institutions, with only a few contributing to the HGP. Examining these publications in a co-authorship network enables us to propose two different sequencing approaches—horizontal and vertical sequencing—whose changing dynamics shaped the history of human genomics. We argue that investigating the extent to which different institutions combined these approaches or prioritized one of them captures the history of genomics better than using the categories of large-scale sequence production and sequence use, as much scholarly literature concerning the HGP has done. Sequence production and use became fully distinct only within the HGP model, and especially during the last stages of this endeavor. By exploring a collaboration between Celera Genomics, a large-scale sequencing institution, and two medical genetics laboratories, we show the potential of our co-authorship network and its analysis for historical research. Our study connects the historiographies of medical genetics and human genomics and indicates that the so-called translational gap from sequence data to clinical outcomes may reflect the assumption that genomics was substantially different from prior and parallel genetics research. This essay is part of a special issue entitled The Sequences and the Sequencers: A New Approach to Investigating the Emergence of Yeast, Human, and Pig Genomics, edited by Miguel García-Sancho and James Lowe.

Relationship analysis related to acid stress of some cornlines used in soil using SSR markings

This research aims to characterize, identify and determine the kinship patterns and bands of genetic diversity in maize lines developed in acid soils from SSRmarkers. This research was carried out at the Molecular Genetics Laboratory, Faculty of Agriculture, Universitas Sumatera Utara, Medan, from March to July 2021. This study used 3 lines and 2 of F1 in corn. To determine band size with the UVITEC Cambridge Fire Reader and to calculate genetic similarity and formation, the group used the Multivariate Statistical Package (MVSP Ver. 3.2). The Research method is the molecular marker method with 3 SSR primers (Cams 358, Tms 7 and Tms 9) the results showed that 3 primers received bands with sizes in the range of 255582 bp. Based on PCOA analysis, there is one sample that dispensed, which equates to 102 713 × NEI 9008. Based on research, CLA 84 × NEI 9008, CLA 106, CLA 84, and NEI9008 are sensitive genotypes and 102 713 × NEI 9008 represent genotypes. tolerant toacid stress.

P.084 5q spinal muscular atrophy Canadian Paediatric Surveillance Program – 2020-2021 results

Background: Spinal muscular atrophy (SMA) is the leading genetic cause of infant death and the second most common autosomal recessive disorder; most cases are due to homozygous deletion of SMN1 gene. Methods: This study uses the Canadian Paediatric Surveillance Program to determine the minimum annual incidence of 5q-SMA from birth to 18 years of age in Canada. The protocol can be accessed at www.cpsp.cps.ca/surveillance. Results: Eighteen cases were reported in 2020-2021. Ten (55%) cases were reported from Ontario and the remaining cases were reported from Atlantic Canada and Western Canada. Their median age was 11 months (IQR 4–21); 61% were male. The most common presenting symptoms were hypotonia and delayed motor milestones in 12 (86%) and 10 (71%) cases respectively. On average, the diagnosis was delayed after onset of symptoms by three months for SMA Type 1, by eight months for Type 2, and by 18 months for Type 3. Twelve (86%) cases received nusinersen as their first disease-modifying treatment. Conclusions: Early recognition and newborn screening are essential to reduce diagnostic delay and enable timely treatment of SMA. Other data sources including the Canadian Neuromuscular Disease Registry and molecular genetic laboratories will be used to estimate the annual incidence of pediatric SMA in Canada.

Evolving fetal phenotypes and clinical impact of progressive prenatal exome sequencing pathways: cohort study.

To determine (1) the diagnostic yield and turnaround time (TAT) of two consecutive prenatal exome sequencing (ES) pathways, (2) the evolution of the fetal phenotype and (3) the clinical impact of detecting causative pathogenic variants and incidental findings. This was a retrospective cohort analysis of prospectively collected fetal cases that underwent trio ES in the presence of a structural anomaly and normal chromosomal microarray testing in the West Midlands Regional Genetics Laboratory, Birmingham, UK. The study included two phases: (1) between July 2018 and October 2020, the clinical pathway from the Prenatal Assessment of Genomes and Exomes (PAGE) study was adopted and involved prenatal trio ES based on a panel of 1542 development disorder genes and case selection by a multidisciplinary team; (2) between October 2020 and July 2021, prenatal trio ES investigation was based on the National Health Service (NHS) England R21 pathway, with definitive inclusion criteria and a panel of 1205 prenatally relevant genes. Deep phenotyping was performed throughout pregnancy and postnatally. A total of 54 cases were included. The diagnostic yield before vs after R21 pathway implementation was 28.0% (7/25) and 55.2% (16/29), respectively (P = 0.04). The respective values for mean TAT were 54.0 days (range, 14-213 days) and 14.2 days (range, 3-29 days). In cases in which a causative pathogenic variant was identified and in which the pregnancy reached the third trimester, additional anomalies were detected between the second and third trimesters in 73.3% (11/15) of cases, predominantly secondary to progressive hydropic features (3/11 (27.3%)), arthrogryposis (3/11 (27.3%)) or brain anomaly (2/11 (18.2%)). In three cases, a variant of uncertain significance was reclassified to likely pathogenic based on postnatal information. Detection of a causative pathogenic variant had a significant clinical impact in 78.3% (18/23) of cases, most frequently affecting decision-making regarding the course of the pregnancy and neonatal management (7/18 (38.9%)). Prenatal ES using the NHS England R21 pathway showed great promise when applied to this cohort, allowing a genetic diagnosis to be made in over half of preselected cases with a fetal structural anomaly on ultrasound. Monitoring and real-time updating of fetal phenotype and reclassification of variants based on postnatal findings is vital to increase the clinical impact that is already evident from this emerging genomic technology. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Genetic diversity analysis of several maize lines (Zea mays L.) developed in acid soil using molecular markers RAPD

Abstact. Maize is the second staple crop in Indonesia with optimal temperature between 21-34 °C, soil pH between 5.6-7.5 and altitude between 1000-1800 m above sea level. Efforts to increase the potential of marginal soils really need to be done to support food needs in Indonesia, one of the ways to be done is by increasing acid stress-resistant maize varieties in Indonesia. This research was conducted at the Molecular Genetics Laboratory of the Faculty of Agriculture University of North Sumatra, Medan from March-July 2021. Determination of band size using the UVITEC Cambridge FireReader software and the calculation of genetic similarities and group formation using the MVSP software (ver. 3.2). Based on 5 plant samples from varietis CIMMYT and Maros then isolated using primers OPC 08, OPD 05 and OPA 03. Based on the results of phylogenetics with UPGMA, all analyzed samples were divided into 2 groups. Group I consistsof CLA 84 × NEI 9008, CLA 84 and NEI 9008. The second group II consisted of CLA 106 and 1027.13 × NEI 9008

Maintenance olaparib in patients (pts) with platinum-sensitive relapsed ovarian cancer (PSROC) by somatic (s) or germline (g) BRCA and other homologous recombination repair (HRR) gene mutation status: Overall survival (OS) results from the ORZORA study.

5519 Background: In the SOLO2 (NCT01874353) trial, maintenance olaparib provided clinically meaningful improvement in OS for PSROC pts with a gBRCA mutation (m) compared with placebo (median 51.7 vs 38.8 months [mo], respectively). The ORZORA trial (NCT02476968) assessed efficacy and safety of maintenance olaparib in PSROC pts with a BRCAm (s or g) or a non-BRCA HRRm. Median progression-free survival (18.0 mo, BRCAm; 16.4, non-BRCA HRRm) was reported at primary data cutoff (DCO). We report final OS analyses. Methods: We conducted an open-label, single-arm, multicenter study of PSROC pts in response to platinum-based chemotherapy (PBC) after ≥2 prior lines of PBC. Pts underwent prospective central screening for tumor BRCAm status (myChoice CDx, Myriad Genetic Laboratories, Inc.), then central gBRCAm testing (BRACAnalysis CDx, Myriad Genetic Laboratories, Inc.) to determine s or g status. An exploratory cohort comprised of pts with predefined non-BRCA HRRm (FoundationOne CDx, Foundation Medicine, Inc.). Pts received maintenance olaparib (400 mg bid; capsules) until progression. OS and time to second progression (PFS2) were secondary endpoints. Results: 181 pts were enrolled (BRCAm n = 145 [s, n = 55; g, n = 87; s/g status unknown, n = 3]; non-BRCA HRRm, n = 33; unassigned, n = 3). At DCO (June 25, 2021), median OS follow-up in censored pts was 42.6 mo in BRCAm and 39.3 mo in non-BRCA HRRm pts. OS and PFS2 are reported in the Table. PBC was received as a subsequent therapy by 33.1% BRCAm, 32.7% sBRCAm, 33.3% gBRCAm, and 45.5% non-BRCA HRRm pts. 177 pts received ≥1 dose of olaparib and were included in safety analyses; 6.2% of pts discontinued because of adverse events (AEs). 37.9% of pts reported grade ≥3 AEs, the most common being anemia (16.4%). Since primary DCO, one new primary malignancy and four myelodysplastic syndrome events occurred. Conclusions: In final OS analyses, maintenance olaparib capsules showed consistent clinical activity in BRCAm and sBRCAm PSROC pts. Exploratory analyses suggest similar activity in non-BRCA HRRm pts. No new safety signals were observed. Findings highlight that PSROC pts, beyond those with a gBRCAm, can benefit from maintenance olaparib. Clinical trial information: NCT02476968. [Table: see text]

THE USE OF MITOCHONDRIAL GENOME POLYMORPHISM TO ESTABLISH PRO-MATERNAL BREEDS IN THE FINAL HYBRIDS OF PIGS

DNA markers are a convenient tool for researching the origin of pig breeds. The characteristic feature of mitochondrial DNA is that mitochondria are transmitted only maternally to all their descendants. Knowing the peculiarity of the transmission of genetic information contained in the mitochondrial DNA, it becomes possible to establish the pro-maternal breeds of pigs of the final hybrid (Large White × Landrace) × Maxgro. Comparison of the maternal-inherited set of rectal fragments of pigs’ final hybrid of foreign selection (n=15) from the total sample (n=175) from RPE "Globinsky Pig Farm", Globyno, town Poltava region, Ukraine allowed getting reliable information of their origin. Laboratory research was carried out based at the Institute of Pig Breeding and Agricultural Production NAAS in the Laboratory of Genetics. As a genetic material, bristles from the auricle of pigs (Large White × Landrace) × Maxgro were used. Allocation was carried out according to Serhii Korinnyi methodology and other authors' publications in 2005 using Chelex -100 ion exchange resin. For the mitochondrial genome analysis, the method of polymorphism of the length of restriction fragments reinforced with amplified in PCR was used (Polymerase chain reaction-restriction fragment of polymorphism – PCR-RFLP). The site of the D-loop of the mitochondrial genome of a pig measuring 428 pairs of nucleotide (with Tas 1 recognition sites in positions 15558, 15580, 15616, 15714, 15758 p.n.) was analyzed. This method allows determining 18 haplotypes of the mitochondrial genome. With its use, mitochondrial haplotypes of pigs of the final hybrid were determined: 4 animals with haplotype C, 6 with haplotype N, and 5 with haplotype O. According to many authors pieces of research, these haplotypes characterize different breeds, namely C – Landrace, N – Large White (Asian type) and O – Landrace. The obtained data on the origin of animals of the final hybrid suggests that two-breed sows were the result of direct (Large White × Landrace) and recipient crossing (Landrace × Large White).It was established that mitochondrial genome polymorphism is an objective marker for determining the breed belonging of animals obtained even in complex hybridization schemes. In our opinion, at the initial stage of breeding work, it is important to establish associations of mitochondrial DNA haplotypes in animals of the final hybrid. Since mitochondria are transmitted along the maternal line only, 13 genes encoding the proteins of the respiratory chain, the identified haplotypes C, N, and O will serve as stable genetic markers. Having determined the correlations of phenotype signs of animals of the final hybrid and certain mitochondrial haplotypes, it is possible to purposefully lead the selection of maternal lines.Genetic examination in the establishment of maternal breeds of hybrid pigs with the help of markers of the mitochondrial genome and the search for a polymorphic area of the X and Y-chromosome to determine the ancestral line has become an urgent issue in continuing our research in modern pig breeding technologies of commercial lines. The work was done with the support of the National Academy of Agrarian Sciences of Ukraine 31.01.00.07. F. “Investigate the pleiotropic effect gens that the SNP use in marker-associated pig breeding”. DR № 0121U109838.

High-Throughput Sequencing of Complementarity Determining Region 3 in the Heavy Chain of B-Cell Receptor in Renal Transplant Recipients: A Preliminary Report.

Background: Graft failure resulting from rejection or any other adverse event usually originates from an aberrant and/or exaggerated immune response and is often catastrophic in renal transplantation. So, it is essential to monitor patients’ immune status for detecting a rejection/graft failure early on. Methods: We monitored the sequence change of complementary determining region 3 (CDR3) in B-cell receptor (BCR) immunoglobulin heavy-chain (IGH) immune repertoire (iR) in 14 renal transplant patients using next-generation sequencing (NGS), correlating its diversity to various clinical events occurring after transplantation. BCR-IGH-CDR3 in peripheral blood mononuclear cells was sequenced along the post-transplantation course by NGS using the iRweb server. Results: Datasets covering VDJ regions of BCR-IGH-CDR3 indicated clonal diversity (D50) variations along the post-transplant course. Furthermore, principal component analysis showed the clustering of these sequence variations. A total of 544 shared sequences were identified before transplantation. D50 remained low in three patients receiving rituximab. Among them, one’s D50 resumed after 3 m, indicating graft tolerance. The D50 rapidly increased after grafting and decreased thereafter in four patients without rejection, decreased in two patients with T-cell-mediated rejection (TCMR) and exhibited a sharp down-sliding after 3 m in two patients receiving donations after cardiac death (DCD). In another two patients with TCMR, D50 was low just before individual episodes, but either became persistently low or returned to a plateau, depending on the failure or success of the immunosuppressive treatments. Shared CDR3 clonal expansions correlated to D50 changes. Agglomerative hierarchical clustering showed a commonly shared CDR3 sequence and at least two different clusters in five patients. Conclusions: Clonal diversity in BCR-IGH-CDR3 varied depending on clinical courses of 14 renal transplant patients, including B-cell suppression therapy, TCMR, DCD, and graft tolerance. Adverse events on renal graft failure might lead to different clustering of BCR iR. However, these preliminary data need further verification in further studies for the possible applications of iR changes as genetic expression biomarkers or laboratory parameters to detect renal graft failure/rejection earlier.

Delayed embryonic development or long sperm survival in embryo donor mares

An embryo collection procedure is usually performed 7-8 days post-ovulation in mares. Embryos are subsequently transferred into a synchronized recipient mare that is expected to carry the pregnancy to term. Submission of a blood or hair root sample to an approved genetics laboratory for DNA parentage verification is a prerequisite for foal registration for most horse breed organizations. The goal of this report is to document two clinical cases in which the DNA parentage test of the foals produced by embryo transfer excluded the stallions whose semen was used on the estrous cycle yielding the embryo and identified as the genetic sire the stallion whose semen was utilized on the previous estrous cycle . Clinical Case # 1. A 21-year-old Arabian mare was inseminated with frozen-thawed semen from a deceased stallion (Stallion A) immediately after detection of a single ovulation. No embryo was recovered following uterine lavage 8 days later. Cooled-transported semen from a different stallion (Stallion B) was used on the subsequent cycle. An exceptionally large (2,466 µm in diameter) expanded blastocyst stage embryo was recovered 8 days post-ovulation and transferred into a recipient mare. The recipient mare carried the pregnancy to term. Genetic testing of the foal excluded Stallion B as the sire and confirmed Stallion A as the genetic sire of the foal. Clinical Case # 2 . A 11-year-old Quarter Horse marewas inseminated with cooled-transported semen from Stallion A and subsequently ovulated two follicles. Uterine lavage 6.5 days later yielded one embryo. Cooled-transported semen from a different stallion (Stallion B), housed at a different reproductive center, was used on the second cycle. The mare ovulated one follicle and uterine lavage 8 days post-ovulation yielded two embryos, one of which was substantially larger than expected. The recipient mare receiving the larger embryo carried the pregnancy to term. DNA parentage testing excluded Stallion B as the genetic sire and confirmed Stallion A as the genetic sire. The intervals from the dates of the original insemination with the genetic sire to the first cycle ovulation, second cycle ovulation and recovery of the embryos in question were 0 days (post-ovulation insemination), 18 days, and 26 days for the Arabian mare and 2 days, 18 days and 26 days for the Quarter Horse mare. The potential explanations for how an equine embryo could be recovered following an insemination 26 days previously include marked delayed fertilization or marked delayed embryonic development. Equine DNA parentage testing relies on the principle of exclusion, with the inheritance of a series of short tandem repeat (STR or microsatellite) markers evaluated in the foal, sire, and embryo donor mare. Parentage assignment based solely on breeding records would have been incorrect.

Optical genome mapping for structural variation analysis in hematologic malignancies.

Optical genome mapping (OGM) is a technology that is rapidly being adopted in clinical genetics laboratories for its ability to detect structural variation (e.g., translocations, inversions, deletions, duplications, etc.) and replace several concurrent standard of care techniques (karyotype, fluorescence in situ hybridization, and chromosomal microarray). OGM can dramatically simplify lab workflow by reducing multiple tests (conventional karyotype, fluorescence in situ hybridization [FISH], and chromosomal microarray) into one test. The superior ability to detect structural variation across the genome removes the need for reflex FISH studies, which can dramatically reduce cost and turnaround time per sample. Parallel studies of OGM versus standard of care testing have demonstrated it can detect and resolve more abnormalities than karyotyping or FISH. However, like many molecular tests that normalize copy number it can have difficulty with non-diploid karyotypes. This Test of the Month review will summarize how the technique works, review the strengths and weaknesses of OGM compared to standard of care techniques and illustrate how the technique is likely to change front line testing in many hematologic malignancies-including summarizing the clinical utility in acute myeloid leukemia, myelodysplastic syndromes, and B cell acute lymphoblastic leukemia.

Simplified floral dip transformation method of Arabidopsis thaliana

Being a model plant, Arabidopsis transformation is a frequent routine in genetics and molecular laboratories. Here, we explain a modified floral dip method routinely used in our laboratory for Arabidopsis transformation by applying drought stress after plant treatment with Agrobacterium. By using this method, hundreds of individual transgenic plants are produced from just 30 infiltrated plants by increasing transformation frequency from 1% to 10% within 2–3 months.

Recommendations for whole genome sequencing in diagnostics for rare diseases

In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.

Mortality Among Patients With Early-Onset Atrial Fibrillation and Rare Variants in Cardiomyopathy and Arrhythmia Genes

Patients with early-onset atrial fibrillation (AF) are enriched for rare variants in cardiomyopathy and arrhythmia genes. The clinical significance of these rare variants in patients with early-onset AF is unknown. To assess the association between rare variants in cardiomyopathy and arrhythmia genes detected in patients with early-onset AF and time to death. This prospective cohort study included participants with AF diagnosed before 66 years of age who underwent whole-genome sequencing through the National Heart, Lung and Blood Institute's Trans-Omics for Precision Medicine program. Participants were enrolled from November 23, 1999, to June 2, 2015. Data were analyzed from February 26 to September 19, 2021. Rare variants identified in a panel of 145 genes that are included in cardiomyopathy and arrhythmia panels used by commercial clinical genetic testing laboratories. The primary study outcome was time to death and was adjudicated from medical records and the National Death Index. Multivariable Cox proportional hazards regression was used to evaluate the association of disease-associated variants with risk of death after adjustment for age at AF diagnosis, sex, race, body mass index, left ventricular ejection fraction, and an interaction term of age at AF diagnosis and disease-associated variant status. Among 1293 participants (934 [72%] male; median age at enrollment, 56.0 years; IQR, 48.0-61.0 years), disease-associated (pathogenic or likely pathogenic) rare variants were found in 131 (10%). During a median follow-up of 9.9 years (IQR, 6.9-13.2 years), 219 participants (17%) died. In univariable analysis, disease-associated variants were associated with an increased risk of mortality (hazard ratio, [HR], 1.5; 95% CI, 1.0-2.1; P = .05); the association remained significant in multivariable modeling when adjusted for age at AF diagnosis, sex, race, body mass index, left ventricular ejection fraction, and an interaction term between disease-associated variant status and age at AF diagnosis. The interaction demonstrated that disease-associated variants were associated with a significantly higher risk of mortality compared with no disease-associated variant when AF was diagnosed at a younger age (P = .008 for interaction). Higher body mass index (per IQR: HR, 1.4; 95% CI, 1.2-1.6; P < .001) and lower left ventricular ejection fraction (per IQR: HR, 0.8; 95% CI, 0.7-0.8; P < .001) were associated with higher mortality risk. There were 73 cardiomyopathy-related deaths, 40 sudden deaths, and 10 stroke-related deaths. Mortality among patients with the most prevalent genes with disease-associated variants was 26% (10 of 38 patients) for TTN, 33% (6 of 18) for MYH7, 22% (2 of 9) for LMNA, 0% (0 of 10) for MYH6, and 0% (0 of 8) for KCNQ1. The findings suggest that rare variants in cardiomyopathy and arrhythmia genes may be associated with increased risk of mortality among patients with early-onset AF, especially those diagnosed at a younger age. Genetic testing may provide important prognostic information for patients with early-onset AF.

Genetics for the pediatric endocrinologists – 1

The advancement in genetic laboratory technology has helped immensely in the diagnosis of many genetic disorders which could not hitherto be diagnosed. Monogenic diabetes among children and adolescents is not uncommon and needs a high index of clinical suspicion to diagnose. With the availability of genetic diagnostic laboratories with the latest technology, more and more patients should benefit from early diagnosis, specific targeted treatment, and better outcomes. The pediatricians and pediatric endocrinologists managing children with diabetes need to clinically suspect and advise appropriate genetic tests to confirm the diagnosis of monogenic diabetes. Neonatal diabetes mellitus is one of the most rewarding diagnoses, if we pick up a specific genetic abnormality that could respond to sulfonylurea. The child with KCNJ11 or ABCC8 gene mutation responding to sulfonylurea could escape from the life-long insulin injections and complications of diabetes. It is equally important to identify other forms of monogenic diabetes as the specific diagnosis can have implications in the treatment, genetic counseling, and identifying other family members harboring the same gene mutation.

A simple method modification to increase separation of 2- and 3-hydroxyglutaric acid by GC–MS for clinical urine organic acids analysis

Urine organic acids profiling by gas chromatography-mass spectrometry (GC–MS) is routinely performed in hospital biochemical genetics laboratories for the investigation of inborn errors of metabolism. In particular, accurate identification of urinary levels of 3-hydroxyglutaric acid (3-OHGA) is important for diagnosing glutaric aciduria type 1 (GA1), but can be challenging by routine GC–MS profiling analysis due to co-elution and spectral similarity with the isomer 2-hydroxyglutaric acid (2-OHGA). To improve analytical specificity, unique ions were selected and a simple second-tier reinjection method was developed to enhance the chromatographic separation of the 2- and 3-OHGA isomers and potential unknown interferences. Specimens flagging on the routine analysis were simply reinjected on the same GC column using a modified temperature gradient containing an isothermal hold. Correlation between the reinjection and initial methods was higher for 2-OHGA (R = 0.9612) compared to 3-OHGA (R = 0.7242). Mean differences between the reinjection and initial methods for 2-OHGA and 3-OHGA were −8.5% and −61.1% respectively. The large decrease in 3-OHGA concentration for many specimens using the reinjection method was primarily attributable to separation from unknown variable interference(s) that were falsely elevating 3-OHGA in the initial analysis despite the use of a more unique quantifier ion. Overall, the reinjection approach increased analytical specificity in evaluating for the presence of increased urinary 3-OHGA. This second-tier approach, using a GC isothermal hold, could easily be implemented or adapted by other clinical laboratories experiencing related diagnostic challenges.

Application Progress of Massively Parallel Sequencing Technology in STR Genetic Marker Detection.

In recent years, more and more forensic genetics laboratories have begun to apply massively parallel sequencing (MPS) technology, that is, next-generation sequencing (NGS) technology, to detect common forensic genetic markers, including short tandem repeat (STR), single nucleotide polymorphism (SNP), the control region or whole genome of mitochondrial DNA (mtDNA), as well as messenger RNA (mRNA), etc., for forensic practice, such as individual identification, kinship analysis, ancestry inference and body fluid identification. As the most widely used genetic marker in forensic genetics, STR is currently mainly detected by capillary electrophoresis (CE) platform. Compared with CE platform, MPS technology has the advantages of simultaneous detection of a large number of genetic markers, massively parallel detection of samples, the polymorphism of sequence detected by NGS makes STR have the advantages of higher resolution and system efficiency. However, MPS technology is expensive, there is no uniform standard so far, and there are problems such as how to integrate MPS-STR data with the existing CE-STR database. This review summarizes the current status of the application of MPS technology in the detection of STR genetic markers in forensic genetics, puts forward the main problems that need to be solved urgently, and prospects the application prospect of this technology in forensic genetics.