Genetic Testing Methodologies Research Articles

Navigating the landscape of clinical genetic testing: insights and challenges in rare disease diagnostics

With the rapid evolution of diagnostic tools, particularly next-generation sequencing, the identification of genetic diseases, predominantly those with pediatric-onset, has significantly advanced. However, this progress presents challenges that span from selecting appropriate tests to the final interpretation of results. This review examines various genetic testing methodologies, each with specific indications and characteristics, emphasizing the importance of selecting the appropriate genetic test in clinical practice, taking into account factors like detection range, cost, turnaround time, and specificity of the clinical diagnosis. Interpretation of variants has become more challenging, often requiring further validation and significant resource allocation. Laboratories primarily classify variants based on the American College of Medical Genetics and Genomics and the Association for Clinical Genomic Science guidelines, however, this process has limitations. This review underscores the critical role of clinicians in matching patient phenotypes with reported genes/variants and considering additional factors such as variable expressivity, disease pleiotropy, and incomplete penetrance. These considerations should be aligned with specific gene-disease characteristics and segregation results based on an extended pedigree. In conclusion, this review aims to enhance understanding of the complexities of clinical genetic testing, advocating for a multidisciplinary approach to ensure accurate diagnosis and effective management of rare genetic diseases.

Current Advances in the Diagnosis and Treatment of Sickle Cell Anaemia

Sickle cell anemia (SCA) presents a complex challenge globally due to its significant morbidity and mortality. Recent advancements in diagnostic tools and treatment modalities have revolutionized the management of this hereditary hemoglobin disorder. This paper aims to synthesize current breakthroughs in the diagnosis and treatment of sickle cell anemia. Diagnostic techniques including genetic testing, hemoglobin electrophoresis, and advanced imaging methodologies are explored, providing insights into their accuracy and clinical applicability. Moreover, novel therapeutic approaches such as gene editing, fetal hemoglobin induction, and targeted therapies are discussed for their potential in ameliorating SCA-related complications and improving patient outcomes. The paper also assesses the challenges and future prospects in the field, emphasizing the need for comprehensive strategies that encompass early detection, personalized interventions, and ongoing research efforts. Understanding these recent strides in both diagnostics and therapeutics is pivotal in enhancing the care and prognosis of individuals afflicted with sickle cell anemia. Keywords: sickle cell anaemia, anaemia, treatment, diagnosis

Medical students' self-perceived knowledge and clinical comfort with genetics in Pakistan.

Pakistan has a high rate of genetic disorders and neonatal mortality concurrent with noted lack of genetic counselors and geneticists. To meet the needs of the patient population, the responsibility of providing clinical genetic services falls on general and specialty physicians. However, their education regarding these essential services is not standardized in medical school curricula nor has it ever been evaluated. The purpose of this work is to describe the self-perceived knowledge, clinical comfort, and perspectives of Pakistani medical students toward their medical genetics' education. A web-based survey was distributed electronically to medical schools around the country. The survey comprised of four sections: (1) participant demographics, (2) self-perceived medical genetics knowledge, (3) level of comfort in applying genetic knowledge and skills, and (4) attitudes toward medical genetics education. Descriptive statistics and a one-way analysis of variance were used for data analysis. Medical students in years 3, 4, and 5 (n = 473) from 25 medical schools participated in this research representing medical education in four Pakistani provinces. Most medical students reported "minimal" to "basic" knowledge of genetic testing methodology (64.7%), cancer genetics (64.9%), prenatal genetic testing (63.02%), and treatment strategies for genetic disease (72.9%). A plurality of students (37%) reported they were uncomfortable with interpreting and communicating genetic test results to patients. Medical students also expressed dissatisfaction with their medical genetics (40%) and genetic counselors training (42%). The self-perceived knowledge and clinical comfort with genetics among Pakistani medical students was limited, especially regarding genetic testing. A significant portion (74.5%) expressed desire for additional genetics education during medical school to aid in their role as future physicians. It is important for physicians-in-training to have a solid understanding of genetic concepts, technologies, and genetic counseling to best support their patients. As endorsed by the participating medical students, this study supports inclusion of more robust genetics' education into Pakistan's medical school curricula.

Spinocerebellar Ataxia Type 35 Caused by a New TGM6 Variant: Video Documentation of a German Family.

Spinocerebellar Ataxia Type 35 Caused by a New TGM6 Variant: Video Documentation of a German Family.

Fryns syndrome: A clinical conundrum

Fryns syndrome is an uncommon autosomal recessive disorder with ocular signs and multisystem involvement with features like diaphragmatic defect, pulmonary hypoplasia, characteristic facial appearance, distal digital hypoplasia, etc., Associated ocular features reported till date are cloudy cornea, microphthalmos, hypertelorism, and retinal dysplasia. The pathogenesis of the syndrome is not well understood although mutations in PIGN gene in clinically diagnosed cases have been reported. Based on a French population study, the prevalence reported was 7 in 100,000 live births, but this prevalence was established before the advent of many genetic testing methodologies. Currently, there are no additional prevalence reports available. We report a 7-month-old female child who presented to us with recurrent acute on chronic dacryocystitis along with microcornea and iridofundal coloboma. Child underwent detailed systemic examination and was clinically diagnosed as Fryns syndrome.

Biallelic SEPSECS variants in two siblings with pontocerebellar hypoplasia type 2D underscore the relevance of splice-disrupting synonymous variants in disease.

Noncoding and synonymous coding variants that exert their effects via alternative splicing are increasingly recognized as an important category of disease-causing variants. In this report, we describe two siblings who presented with hypotonia, profound developmental delays, and seizures. Brain magnetic resonance imaging (MRI) in the proband at 5 yr showed diffuse cerebral and cerebellar white matter volume loss. Both siblings later developed ventilator-dependent respiratory insufficiency and scoliosis and are currently nonverbal and nonambulatory. Extensive molecular testing including oligo array and clinical exome sequencing was nondiagnostic. Research genome sequencing under an institutional review board (IRB)-approved study protocol revealed that both affected children were compound-heterozygous for variants in the SEPSECS gene. One variant was an initiator codon change (c.1A > T) that disrupted protein translation, consistent with the observation that most disease-causing variants are loss-of-function changes. The other variant was a coding change (c.846G > A) that was predicted to be synonymous but had been demonstrated to disrupt mRNA splicing in a minigene assay. The SEPSECS gene encodes O-phosphoseryl-tRNA(Sec) selenium transferase, an enzyme that participates in the biosynthesis and transport of selenoproteins in the body. Variations in SEPSECS cause autosomal recessive pontocerebellar hypoplasia type 2D (PCHT 2D; OMIM #613811), a neurodegenerative condition characterized by progressive cerebrocerebellar atrophy, microcephaly, and epileptic encephalopathy. The identification of biallelic pathogenic variants in this family—one of which was a synonymous change not identified by prior clinical testing—not only ended the diagnostic odyssey for this family but also highlights the contribution of occult pathogenic variants that may not be recognized by standard genetic testing methodologies.

Utilization of genetic testing: Analysis of 4,499 prior authorization requests for molecular genetic tests at four US regional health plans.

Continuous advances in genetic testing methodologies and an increased understanding of the genetic mechanisms of diseases have fueled genetic testing utilization across health care specialties. To our knowledge, national trends in the ordering of genetic testing have not been studied broadly across clinical indications, testing methodologies, and ordering provider specialties. We performed a retrospective analysis of 4,499 complete prior authorization requests for molecular genetic testing submitted to four regional health plans' commercial lines of business between May 1, 2019 and May 31, 2019. Ordering providers were characterized by their certification(s) and specialty of practice. Among 4,499 genetic testing requests, 92% were ordered by non-genetics providers. Obstetrician/gynecology (OBGYN) (63%), oncology (15%), and genetics (8%) providers ordered genetic testing most frequently. Reproductive, hereditary cancer, and tumor testing were the most frequently ordered genetic tests. Seventy-nine percent of all prior authorization requests were approved. When analyzing complex genetic testing requests, we found that testing ordered by genetics providers was more likely to be approved based on health plan policy than testing ordered by non-genetics providers. Our results suggest that health care providers across multiple medical specialties may benefit from involvement of genetics specialists in decision-making regarding molecular tests.

Evaluation of the results of patients who applied to the Çukurova University, Medical Genetics Department for prenatal diagnosis and determination of genetic counseling principles

Background/aim The aim of this study was to summarize the experiences of a single medical center for genetic diagnosis and treatment of prenatal patients.Materials and methods This study includes a retrospective data analysis of 2843 prenatally investigated cases using invasive methods during a 6-year period (2013–2019) at a single tertiary care center.Results Chromosomal abnormalities were detected in 80 out of 1221 amniotic fluid samples;,178 out of 1608 chorionic villus samples, and 1 out of 14 cordocentesis samples. The most common chromosomal abnormality was trisomy 21. At least one mutation was detected in 63 of the 152 molecular tests performed on fetuses. Conclusion Clinical procedures such as ultrasounds and genetic tests are able to provide a better clinical follow-up for pregnant women about the possible congenital anomalies or any genetic condition, with proper genetic counseling and testing methodology.

Making a Genetic Diagnosis in a Level IV Neonatal Intensive Care Unit Population: Who, When, How, and at What Cost?

To investigate the prevalence of genetic disease and its economic impact in a level IV neonatal intensive care unit (NICU) by identifying and describing diseases diagnosed, genetic testing methodologies used, timing of diagnosis, length of NICU stay, and charges for NICU care. A retrospective chart review of patients admitted to a level IV NICU from 2013 to 2014 (n=1327) was undertaken and data collected up to 2years of age from the electronic medical record. In total, 117 patients (9%) received 120 genetic diagnoses using a variety of methodologies. A significant minority of diagnoses, 36%, were made after NICU discharge and 41% were made after 28days of age. Patients receiving a genetic diagnosis had significantly longer mean lengths of stay (46days vs 29.1days; P<.01) and costlier mean charges ($598 712 vs $352 102; P<.01) for their NICU care. The NICU stay charge difference to care for a newborn with a genetic condition was on average $246 610 in excess of that for a patient without a genetic diagnosis, resulting in more than $28 000 000 in excess charges to care for all patients with genetic conditions in a single NICU over a 2-year period. Given the high prevalence of genetic disease in this population and the documented higher cost of care, shortening the time to diagnosis and targeting therapeutic interventions for this population could make a significant impact on neonatal care in level IV NICUs.

Genetic Influences on Behavioral Outcomes After Childhood TBI: A Novel Systems Biology-Informed Approach.

Objectives: To test whether genetic associations with behavioral outcomes after early childhood traumatic brain injury (TBI) are enriched for biologic pathways underpinning neurocognitive and behavioral networks.Design: Cross-sectional evaluation of the association of genetic factors with early (~ 6 months) and long-term (~ 7 years) post-TBI behavioral outcomes. We combined systems biology and genetic association testing methodologies to identify biologic pathways associated with neurocognitive and behavior outcomes after TBI. We then evaluated whether genes/single nucleotide polymorphism (SNPs) associated with these biologic pathways were more likely to demonstrate a relationship (i.e., enrichment) with short and long-term behavioral outcomes after early childhood TBI compared to genes/SNPs not associated with these biologic pathways.Setting: Outpatient research setting.Participants:140 children, ages 3–6:11 years at time of injury, admitted for a TBI or orthopedic injury (OI).Interventions: Not Applicable.Main Outcome Measures: Child behavior checklist total problems T score.Results: Systems biology methodology identified neuronal systems and neurotransmitter signaling (Glutamate receptor, dopamine, serotonin, and calcium signaling), inflammatory response, cell death, immune systems, and brain development as important biologic pathways to neurocognitive and behavioral outcomes after TBI. At 6 months post injury, the group (TBI versus OI) by polymorphism interaction was significant when the aggregate signal from the highest ranked 40% of case gene associations was compared to the control set of genes. At ~ 7 years post injury, the selected polymorphisms had a significant main effect after controlling for injury type when the aggregate signal from the highest ranked 10% of the case genes were compared to the control set of genesConclusions: Findings demonstrate the promise of applying a genomics approach, informed by systems biology, to understanding behavioral recovery after pediatric TBI. A mixture of biologic pathways and processes are associated with behavioral recovery, specifically genes associated with cell death, inflammatory response, neurotransmitter signaling, and brain development. These results provide insights into the complex biology of TBI recovery.

Molecular genetic testing methodologies in hematopoietic diseases: current and future methods.

Rapid technological advancements in clinical molecular genetics have increased our diagnostic and prognostic capabilities in health care. Understanding these assays, as well as how they may change over time, is critical for pathologists, clinicians, and translational researchers alike. This review provides a practical summary and basic reference for current molecular genetic technologies, as well as new testing methodologies that are in use, gaining momentum, or anticipated to contribute more broadly in the future. Here, we discuss DNA and RNA based methodologies including classic assays such as the polymerase chain reaction (PCR), Sanger sequencing, and microarrays, to more cutting-edge next-generation sequencing (NGS) based assays and emerging molecular technologies such as cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA), and NGS-based detection of infectious disease organisms. This review serves as a basic foundation for knowledge in current and emerging clinical molecular genetic technologies.

Recommendations for the clinical interpretation of genetic variants and presentation of results to patients with inherited bleeding disorders. A UK Haemophilia Centre Doctors' Organisation Good Practice Paper.

This paper sets out good practice for clinicians involved in interpreting variant reports for patients with inherited bleeding disorders. It is aimed primarily at doctors, nurses and allied healthcare professionals who may not have had specific training in genetic testing methodology or reporting. It deals with uncertainty in classification of variant pathogenicity and the handling of incidental findings.

The changing scenario in prenatal diagnosis of genetic disorders: Genetics to genomics

BackgroundPrevention of genetic disorders in the family is gaining significance because of the burden of these untreatable and devastating disorders. Sir Ganga Ram Hospital (SGRH) has been at the forefront for prenatal diagnosis (PND) in India for the last 2 decades. Aims and objectivesEvaluation of pregnant women for prenatal diagnosis in year 2017, and comparison with the centre's historical 5-year data published in 2003. Material and methodsPregnant women attending genetic clinics at SGRH and prenatal samples received from elsewhere were enrolled. Cytogenetic, molecular, biochemical and non-invasive methods were utilized for PND. ResultsAmong 3351 pregnant women from SGRH, 2530 presented with obstetric indications, and 821 women had a background family history. In cytogenetic laboratory, 706 prenatal samples from SGRH and 553 samples from elsewhere were analyzed. Additional 623 women opted for Non-invasive prenatal test (NIPT) for aneuploidies. In molecular genetics laboratory, 733 prenatal samples were analyzed. Highlights in the study included the introduction of NIPT, and other major advances - chromosomal microarrays and next-generation sequencing enabling PND in 130 and 20 cases, respectively. NIPT detected 10 cases of aneuploidy, with 10% false positivity in 623 women. Comparison with 5-year data from SGRH revealed a 6–10 fold rise in number of PNDs being performed, and addition of newer indications, materialized by newer methods of NGS and CMAs. ConclusionAdvanced genetic testing methodologies like NIPT, NGS and CMA are rapidly changing the scenario of prenatal diagnosis in our country, which is reflected in our data of last one year.

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.242Neonatal presentations of recessive TTN-related myopathy: an emerging distinct clinical phenotype

With the increasing availability of next generation-based sequencing, the phenotypic spectrum associated with TTN-related myopathies is ever-expanding. Early clinical recognition is essential to facilitate accurate genetic testing methodologies, to aid in pathogenicity interpretation of the large number of TTN variants identified, and to direct proper medical care and management. Here we report four unrelated patients, aged 2 to 12 years, with genetically confirmed biallelic TTN mutations who display a consistent, recurrent phenotype that is recognizable at birth. Retrospective review of the neonatal presentations of these four patients revealed characteristic clinical features, the most prominent of which is striking positioning of the hands and feet, namely bilateral wrist extension with ulnar deviation of the hands and extreme ankle dorsiflexion of the feet with associated Achilles tendon hyperlaxity. Additional salient features include reduced fetal movements and oligohydramnios, along with bulbar weakness clinically manifesting as dysphagia and feeding difficulties, generalized hypotonia and joint hypermobility. Bilateral hip dysplasia and respiratory distress or apnea was also seen in three out of four neonates. This cohort delineates a distinct and recognizable neonatal clinical phenotype associated with recessive TTN-related myopathy and may aid in the recognition of titinopathy in neonates presenting with features of a congenital myopathy. It can be added as a ``titin-compatible'' phenotype that will also help in interpreting TTN genotypes ``of uncertain significance.'' Additional neonatal titinopathy cases will surely expand on this phenotype. Longitudinal data are needed to further characterize this phenotype and increase our understanding of the natural history of titinopathy as well as expand genotype-phenotype correlations.

A comparison of genetic sampling methodologies for candidate-gene analyses.

Much of the recent gains in knowledge regarding the influence of patient genetics on medication pharmacokinetics (drug absorption, distribution, metabolism and elimination) how patients process medications) and pharmacodynamics (drug response) have been attributed to the technologic advances in genetic testing methodologies and the involvement of large clinical data sets and biobanks. Indeed, Genome Wide Association Studies (GWAS) and Phenome Wide Association Studies (PWAS) along with ever-evolving biomedical informatics techniques and the expansion of the -omics sciences (e.g., transcriptomics, metabolomics, proteomics) have brought about unprecedented advances in precision medicine. Although the simpler candidate-gene analysis technique is not considered cutting-edge, it is reliable and important to the translation of pharmacogenomic research and the advancement of precision medicine. Leveraging the knowledge of biological plausibility (i.e., genetic mutation → altered function of protein product → altered drug pharmacokinetics/dynamics) to appropriately select genes for inclusion, the candidate-gene analysis technique does not necessitate large patient cohorts nor extensive multi-gene genetic analysis arrays. It is often the ideal method for clinicians to begin evaluating whether genetic information might improve their pharmacologic treatment strategies for their patients. Having access to specific patient populations and expertise regarding their medical subspecialty, physician scientists can implement a candidate-gene analysis in small cohorts. Even with less than 100 patients, results can often be used to determine whether further investigation is warranted and to inform future studies. Herein, we present a comparison of select contemporary methodologies regarding collection, processing and genotype testing applicable to the efficient implementation of candidate-gene studies.

Genetic Testing of Maturity-Onset Diabetes of the Young Current Status and Future Perspectives.

Diabetes is a global epidemic problem growing exponentially in Asian countries posing a serious threat. Among diabetes, maturity-onset diabetes of the young (MODY) is a heterogeneous group of monogenic disorders that occurs due to β cell dysfunction. Genetic defects in the pancreatic β-cells result in the decrease of insulin production required for glucose utilization thereby lead to early-onset diabetes (often <25 years). It is generally considered as non-insulin dependent form of diabetes and comprises of 1–5% of total diabetes. Till date, 14 genes have been identified and mutation in them may lead to MODY. Different genetic testing methodologies like linkage analysis, restriction fragment length polymorphism, and DNA sequencing are used for the accurate and correct investigation of gene mutations associated with MODY. The next-generation sequencing has emerged as one of the most promising and effective tools to identify novel mutated genes related to MODY. Diagnosis of MODY is mainly relying on the sequential screening of the three marker genes like hepatocyte nuclear factor 1 alpha (HNF1α), hepatocyte nuclear factor 4 alpha (HNF4α), and glucokinase (GCK). Interestingly, MODY patients can be managed by diet alone for many years and may also require minimal doses of sulfonylureas. The primary objective of this article is to provide a review on current status of MODY, its prevalence, genetic testing/diagnosis, possible treatment, and future perspective.

Chapter 2 - Genetic and genomic testing for neurologic disease in clinical practice

The influence of genetics on neurologic disease is broad and it is becoming more common that clinicians are presented with a patient whose disease is likely of genetic origin. In the search for mutations causing Mendelian disorders, advances in genetic testing methodology have propelled modern neurologic practice beyond single-gene testing into the realm of genomic medicine, where routine evaluations encompass hundreds or thousands of genes, or even the entire exome, representing all protein-coding genes in the genome. The role of various single-gene, multigene, and genomic testing methods, including chromosomal microarray and next-generation sequencing, in the evaluation of neurologic disease is discussed here to provide a framework for their use in a modern neurologic practice. Understanding the inherent issues that arise during the interpretation of sequence variants as pathogenic or benign and the potential discovery of incidental medically relevant findings are important considerations for neurologists utilizing these tests clinically. Strategies for the evaluation of clinically heterogeneous disorders are presented to guide neurologists in the transition from single-gene to genomic considerations and toward the prospect of the widespread routine use of exome sequencing in the continuing goal to achieve more rapid and more precise diagnoses that will improve management and outcome in patients challenged by neurologic disease.

The utility of genetic testing in neuromuscular disease: A consensus statement from the AANEM on the clinical utility of genetic testing in diagnosis of neuromuscular disease.

The aim of this consensus statement is to provide a recommendation from AANEM experts on the clinical utility of genetic testing. It is not meant to recommend or endorse any specific genetic testing methodology or algorithm. The AANEM Professional Practice Committee reached a consensus based on expert opinion on the utility of genetic testing in neuromuscular diseases and made recommendations on factors that physicians and patients should consider when deciding whether to proceed with such testing. Despite the costs of genetic testing, these tests can be both valuable and beneficial in the diagnosis and treatment of neuromuscular diseases in many situations. The AANEM believes that performing genetic testing to arrive at a specific molecular diagnosis is a critical step in providing high-quality care to neuromuscular patients. The cost of testing should not be a deterrent, as there are important clinical, safety, psychosocial, and research benefits. Muscle Nerve 54: 1007-1009, 2016.

Challenges in Molecular Diagnostics of Channelopathies in the Next-Generation Sequencing Era: Less Is More?

Inherited arrhythmogenic diseases (IADs – also called cardiac channelopathies) are defined as a group of genetic diseases characterized by electrically unstable substrate in a structurally normal heart (1). Genetic testing in cardiac channelopathies has completed its transition from a research-based activity to that of a clinical genetic service. In parallel, the advancements of the sequencing technologies are providing ways to sequence several genes at a relatively low cost. This is progressively changing the approach to the genetic diagnosis of IADs. Indeed, while Sanger-based genetic testing was traditionally limited to the well characterized, most prevalent genes, next-generation sequencing (NGS) allows screening even the “minor” disease genes with very short turnaround time. This approach to genetic testing is highly efficient, but it is also generating remarkable interpretative problems mostly related to the high prevalence of variants of unknown significance (VUS), i.e., not clearly related to disease pathophysiology. This issue is relevant to several genetic diseases of the heart but is particularly evident in IADs, where the familial co-segregation analysis is hampered by the incomplete penetrance and the variable expressivity. How to design the most appropriate screening approach is challenging and it requires in-depth knowledge of the specific diseases of interest. In this Opinion article, we will review the available NGS approaches and try to outline the available strategies to optimize the performance of this genetic testing methodology.

Genetic tests obtainable through pharmacies: the good, the bad, and the ugly

Genomic medicine seeks to exploit an individual’s genomic information in the context of guiding the clinical decision-making process. In the post-genomic era, a range of novel molecular genetic testing methodologies have emerged, allowing the genetic testing industry to grow at a very rapid pace. As a consequence, a considerable number of different private diagnostic testing laboratories now provide a wide variety of genetic testing services, often employing a direct-to-consumer (DTC) business model to identify mutations underlying (or associated with) common Mendelian disorders, to individualize drug response, to attempt to determine an individual’s risk of a multitude of complex (multifactorial) diseases, or even to determine a person’s identity. Recently, we have noted a novel trend in the provision of private molecular genetic testing services, namely saliva and buccal swab collection kits (for deoxyribonucleic acid (DNA) isolation) being offered for sale over the counter by pharmacies. This situation is somewhat different from the standard DTC genetic testing model, since pharmacists are healthcare professionals who are supposedly qualified to give appropriate advice to their clients. There are, however, a number of issues to be addressed in relation to the marketing of DNA collection kits for genetic testing through pharmacies, namely a requirement for regulatory clearance, the comparative lack of appropriate genetics education of the healthcare professionals involved, and most importantly, the lack of awareness on the part of both the patients and the general public with respect to the potential benefits or otherwise of the various types of genetic test offered, which may result in confusion as to which test could be beneficial in their own particular case. We believe that some form of genetic counseling should ideally be integrated into, and made inseparable from, the genetic testing process, while pharmacists should be obliged to receive some basic training about the genetic tests that they offer for sale.