Precise Molecular Diagnosis Research Articles

Three Novel Variants identified in FBN1 and TGFBR2 in seven Iranian families with suspected Marfan syndrome.

BackgroundMarfan syndrome (MFS) is a multi‐systemic autosomal dominant disease of the connective tissue characterized by the early development of thoracic aneurysms/dissections, along with various manifestations of the ocular and skeletal systems. Due to the genetic and clinical heterogeneity, the clinical diagnosis of this disorder is challenging. Loss‐of‐function mutations in FBN1 (encodes fibrillin‐1) lead to MFS type 1. Also, similar mutations in transforming growth factor β receptor 2 (TGFBR2) gene cause MFS type 2. Both proteins involve in TGF‐β signaling.MethodsIn this study, genetic screening using a panel involving 14 genes, especially FBN1 and TGFBR2, were performed on seven representatives affected members of seven unrelated Iranian families suspected with MFS. To confirm the variants, Sanger sequencing was applied to other affected/unaffected members of the families.ResultsA total of 13 patients showed MFS manifestations. Using genetic screening, two novel and three previously reported variants in FBN1 were identified. We also detected two variants (a novel and a previously reported variant) in the TGFBR2 gene.ConclusionIn this study, we introduce three novel variants identified through gene screening in seven Iranian MFS families. This report is expected to considerably improve genetic counseling for Iranian MFS families. Early precise molecular diagnosis can be helpful for better management and improving the life expectancy of these patients.

Genetic Sequencing of Pediatric Patients Identifies Mutations in Monogenic Inflammatory Bowel Disease Genes that Translate to Distinct Clinical Phenotypes.

OBJECTIVES:Monogenic inflammatory bowel disease (IBD) comprises rare Mendelian causes of gut inflammation, often presenting in infants with severe and atypical disease. This study aimed to identify clinically relevant variants within 68 monogenic IBD genes in an unselected pediatric IBD cohort.METHODS:Whole exome sequencing was performed on patients with pediatric-onset disease. Variants fulfilling the American College of Medical Genetics criteria as “pathogenic” or “likely pathogenic” were assessed against phenotype at diagnosis and follow-up. Individual patient variants were assessed and processed to generate a per-gene, per-individual, deleteriousness score.RESULTS:Four hundred one patients were included, and the median age of disease-onset was 11.92 years. In total, 11.5% of patients harbored a monogenic variant. TRIM22-related disease was implicated in 5 patients. A pathogenic mutation in the Wiskott-Aldrich syndrome (WAS) gene was confirmed in 2 male children with severe pancolonic inflammation and primary sclerosing cholangitis. In total, 7.3% of patients with Crohn's disease had apparent autosomal recessive, monogenic NOD2-related disease. Compared with non-NOD2 Crohn's disease, these patients had a marked stricturing phenotype (odds ratio 11.52, significant after correction for disease location) and had undergone significantly more intestinal resections (odds ratio 10.75). Variants in ADA, FERMT1, and LRBA did not meet the criteria for monogenic disease in any patients; however, case-control analysis of mutation burden significantly implicated these genes in disease etiology.DISCUSSION:Routine whole exome sequencing in pediatric patients with IBD results in a precise molecular diagnosis for a subset of patients with IBD, providing the opportunity to personalize therapy. NOD2 status informs risk of stricturing disease requiring surgery, allowing clinicians to direct prognosis and intervention.

Stimulus-responsive surface-enhanced Raman scattering: a "Trojan horse" strategy for precision molecular diagnosis of cancer.

Molecular diagnosis has played an increasingly important role in cancer detection. However, it remains challenging to develop an in situ analytical method capable of profiling the molecular phenotype of tumors for precision cancer diagnosis. A “Trojan horse” strategy based on stimulus-responsive surface-enhanced Raman scattering (SR-SERS) is reported here for selectively recording the comprehensive molecular information of tumors in situ, without resorting to destructive sample preparation and complex data analysis. This technique is employed to delineate the margin between tumors and normal tissues with high accuracy, and to further discriminate the molecular fingerprints of tumors in the early and late stages. Based on molecular profiling, we discovered that the signal ratios of fatty acid-to-phenylalanine could serve as promising indicators for identifying the primary tumors in different stages. This simple SR-SERS technique also provides a potential useful means for identifying tumor classifications or distinguishing primary and metastatic tumors.

Ambient mass spectrometry for the molecular diagnosis of lung cancer.

Lung cancer is one of the most common malignancies and the leading cause of cancer-related death worldwide. Among the technologies suitable for the rapid and accurate molecular diagnosis of lung cancer, ambient mass spectrometry (AMS) has gained increasing interest as it allows the direct profiling of molecular information from various biological samples (e.g., tissue, serum, urine and sputum) in real-time and with minimal or no sample pretreatment. This minireview summarizes the applications of AMS in lung cancer studies (including tissue molecular identification, the discovery of potential biomarkers, and surgical margin assessment), and discusses the challenges and perspectives of AMS in the clinical precision molecular diagnosis of lung cancer.

Sequential Detection of Lipids, Metabolites, and Proteins in One Tissue for Improved Cancer Differentiation Accuracy.

Traditionally, molecular information on metabolites, lipids, and proteins is collected from separate individual tissue samples using different analytical approaches. Herein a novel strategy to minimize the potential material losses and the mismatch between metabolomics, lipidomics, and proteomics data has been demonstrated based on internal extractive electrospray ionization mass spectrometry (iEESI-MS). Sequential detection of lipids, metabolites, and proteins from the same tissue sample was achieved without sample reloading and hardware alteration to MS instrument by sequentially using extraction solutions with different chemical compositions. With respect to the individual compound class analysis, the sensitivity, specificity, and accuracy obtained with the integrative information on metabolites, lipids, and proteins from 57 samples of 13 patients for lung cancer prediction was substantially improved from 54.0%, 51.0%, and 76.0% to 100.0%, respectively. The established method is featured by low sample consumption (ca. 2.0 mg) and easy operation, which is important to minimize systematic errors in precision molecular diagnosis and systems biology studies.

Atypical cerebral palsy: genomics analysis enables precision medicine

PurposeThe presentation and etiology of cerebral palsy (CP) are heterogeneous. Diagnostic evaluation can be a prolonged and expensive process that might remain inconclusive. This study aimed to determine the diagnostic yield and impact on management of next-generation sequencing (NGS) in 50 individuals with atypical CP (ACP). MethodsPatient eligibility criteria included impaired motor function with onset at birth or within the first year of life, and one or more of the following: severe intellectual disability, progressive neurological deterioration, other abnormalities on neurological examination, multiorgan disease, congenital anomalies outside of the central nervous system, an abnormal neurotransmitter profile, family history, brain imaging findings not typical for cerebral palsy. Previous assessment by a neurologist and/or clinical geneticist, including biochemical testing, neuroimaging, and chromosomal microarray, did not yield an etiologic diagnosis. ResultsA precise molecular diagnosis was established in 65% of the 50 patients. We also identified candidate disease genes without a current OMIM disease designation. Targeted intervention was enabled in eight families (~15%). ConclusionNGS enabled a molecular diagnosis in ACP cases, ending the diagnostic odyssey, improving genetic counseling and personalized management, all in all enhancing precision medicine practices.

Maturity onset diabetes of the young (MODY) in Chinese children: genes and clinical phenotypes.

Background To investigate the clinical and molecular characteristics of Chinese children with maturity onset diabetes of the young (MODY). Methods A total of 42 Chinese patients suspected MODY referred to our unit from 2014 to 2018 were enrolled. Mutational analysis of monogenic diabetes mellitus genes was performed by next-generation sequencing and confirmed by Sanger sequencing. Results There were 28 males (66.7%) and 14 females (33.3%) with a mean age of 9.49 ± 3.46 years (range, 1.4-15.3 years) and a mean birth weight of 3.38 ± 0.49 kg (range, 2.55-4.90 kg). Among these patients, 15 patients had polyuria, polydipsia or weight loss. Two patients (4.8%) were obese and six (14.3%) were overweight. Moreover, 13 patients (30.9%) had a family history of diabetes. Thirty variants were identified in 28 patients. Twenty-six variants in 25 patients were pathogenic or likely pathogenic genes (59.5%, 25/42), including 15 patients (60.0%, 15/25) with GCK mutation, four (16.0%, 4/25) with PAX4 mutation, three (12.0%, 3/25) with HNF4A mutation, one (4.0%, 1/25) with INS mutation, one (4.0%, 1/25) with NEUROD1 mutation and one (4.0%, 1/25) with HNF1A mutation. Nine mutations (36.0%, 9/25) were novel. There was no difference between mutation-suspected patients and MODY-confirmed patients except for a 2-h glucose increment in an oral glucose tolerance test (OGTT), while the GCK-MODY had lower glycated hemoglobin (HbA1c) and a significantly smaller 2-h glucose increment in an OGTT compared with transcription factor MODYs. The GCK-MODY was identified by incidental hyperglycemia without glycosuria. GCK-MODY without drug management and hepatocyte nuclear factor-1 alpha (HNF4A) or HNF1A-MODY with sulfonylurea therapy obtained good glucose controlling. Conclusions Mutation of the GCK gene is the most common in MODY patients in China followed by PAX4. The screening criteria can improve the cost-effectiveness of disease diagnosis and treatment. A precise molecular diagnosis would lead to optimal treatment of the patients.

BBS5 and INPP5E mutations associated with ciliopathy disorders in families from Pakistan.

Ciliopathies are a clinically and genetically heterogeneous group of disorders often exhibiting phenotypic overlap and caused by abnormalities in the structure or function of cellular cilia. As such, a precise molecular diagnosis is important for guiding clinical management and genetic counseling. In the present study, two Pakistani families comprising individuals with overlapping clinical features suggestive of a ciliopathy syndrome, including intellectual disability, obesity, congenital retinal dystrophy, and hypogonadism (in males), were investigated clinically and genetically. Whole-exome sequencing identified the likely causes of disease as a novel homozygous frameshift mutation (NM_152384.2: c.196delA; p.(Arg66Glufs*12); family 1) in BBS5, and a nonsense mutation (NM_019892.5:c.1879C>T; p.Gln627*; family 2) in INPP5E, previously reported in an extended Pakistani family with MORM syndrome. Our findings expand the molecular spectrum associated with BBS5 mutations in Pakistan and provide further supportive evidence that the INPP5E mutation is a common cause of ciliopathy in Northern Pakistan, likely representing a regional founder mutation. This study also highlights the value of genomic studies in Pakistan for families affected by rare heterogeneous developmental disorders and where clinical phenotyping may be limited by geographical and financial constraints. The identification of the spectrum and frequency of disease-causing variants within this setting enables the development of population-specific genetic testing strategies targeting variants common to the local population and improving health care outcomes.

Copy number variant and runs of homozygosity detection by microarrays enabled more precise molecular diagnoses in 11,020 clinical exome cases

BackgroundExome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES.MethodsIn a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array.ResultsThe QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities.ConclusionsOur clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations.

Mutational spectrum of dystrophinopathies in Singapore: Insights for genetic diagnosis and precision therapy.

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive disorders caused by mutations in the DMD gene. Emerging therapies targeting patients with specific mutations are now becoming a reality for many of these patients. Precise molecular diagnosis is essential to facilitate the identification of possible new treatments for patients in the local context. In this study, we screened 145 dystrophinopathic patients in Singapore and assessed their molecular status for eligibility to current emerging genetic therapies. Overall, 140 (96.5%) of all patients harbored pathogenic DMD mutations comprising 95 exonic deletions (65.5%), 14 exonic duplications (9.7%), and 31 pathogenic small mutations (21.4%). Nonsense and frameshift mutations constitute 83.9% of all the small mutations. We found 71% (103/145) of all Singaporean dystrophinopathy patients to be theoretically amenable for exon skipping, either through skipping of single (53.1%) or multiple exons (17.9%). This approach is applicable to 81.1% (77/95) of patients carrying deletions and 83.9% (26/31) of those with small mutations. Eteplirsen induced skipping of exon 51 is applicable to 12.4% of local patients. Nonsense read-through therapy was found to be applicable in another 12.4% of all patients. Mutation screening is crucial for providing insights into the underlying genetic signature of the disease in the local population and contributes toward existing information on DMD mutations in Asia and globally. This will guide future targeted drug development and clinical trial planning for this disease.

Collagen stiffness promoted non-muscle-invasive bladder cancer progression to muscle-invasive bladder cancer.

Purpose: Bladder cancer (BCa) is generally considered one of the most prevalent deadly diseases worldwide. Patients suffering from muscle-invasive bladder cancer (MIBC) possess dismal prognoses, while those with non-muscle-invasive bladder cancer (NMIBC) generally have a favorable outcome after local treatment. However, some NMIBCs relapse and progress to MIBC, with an unclarified mechanism. Hence, insight into the genetic drivers of BCa progression has tremendous potential benefits for precision therapeutics, risk stratification, and molecular diagnosis.Methods: In this study, three cohorts profile datasets (GSE13507, GSE32584, and GSE89) consisting of NMIBC and MIBC samples were integrated to address the differently expressed genes (DEGs). Subsequently, the protein-protein interaction (PPI) network and pathway enrichment analysis of DGEs were performed.Results: Six collagen members (COL1A1, COL1A2, COL5A2, COL6A1, COL6A2, and COL6A3) were up-regulated and gathered in the ECM-receptor interaction signal pathway identified by KEGG pathway analysis and GSEA. Evidence derived from the Oncomine and TCGA databases indicated that the 6 collagen genes promote the progression of BCa and are negatively associated with patient prognosis. Moreover, taking COL1A1 as a further research object, the results showed that COL1A1 was up-regulated in MIBC and its knockdown significantly inhibited the proliferation, migration, and invasion of 5637 and T24 cells by inhibiting epithelial-mesenchymal transition (EMT) process and the TGF-β signaling pathway.Conclusion: With integrated bioinformatic analysis and cell experiments, we showed that 6 collagen family members are high progression risk factors and that they can be used as independent effective diagnostic and prognostic biomarkers for BCa.

Recent progress in laboratory diagnosis of thalassemia and hemoglobinopathy: a study by the Korean Red Blood Cell Disorder Working Party of the Korean Society of Hematology.

Genetic hemoglobin disorders are caused by mutations and/or deletions in the α-globin or β-globin genes. Thalassemia is caused by quantitative defects and hemoglobinopathies by structural defect of hemoglobin. The incidence of thalassemia and hemoglobinopathy is increased in Korea with rapid influx of people from endemic areas. Thus, the awareness of the disease is needed. α-thalassemias are caused by deletions in α-globin gene, while β-thalassemias are associated with decreased synthesis of β-globin due to β-globin gene mutations. Hemoglobinopathies involve structural defects in hemoglobin due to altered amino acid sequence in the α- or β-globin chains. When the patient is suspected with thalassemia/hemoglobinopathy from abnormal complete blood count findings and/or family history, the next step is detecting hemoglobin abnormality using electrophoresis methods including high performance liquid chromatography and mass spectrometry. The development of novel molecular genetic technologies, such as massively parallel sequencing, facilitates a more precise molecular diagnosis of thalassemia/hemoglobinopathy. Moreover, prenatal diagnosis using genetic testing enables the prevention of thalassemia birth and pregnancy complications. We aimed to review the spectrum and classification of thalassemia/hemoglobinopathy diseases and the diagnostic strategies including screening tests, molecular genetic tests, and prenatal diagnosis.

Outcome and factors affecting survival of childhood myelodysplastic syndrome; single centre experience

Abstract Introduction Although many therapeutic options have been attempted for pediatric myelodysplastic syndrome (MDS), still hematopoietic stem cell transplant (HSCT) is the only curative therapy. We described the outcome of these patients and the factors affecting the overall survival (OS) and event free survival (EFS). Methods This is a retrospective descriptive study included 44 patients with MDS who presented to Children Cancer Hospital Egypt (CCHE-57357), between July 2007 and December 2015. HSCT was offered to patients who had HLA matched family donors. Results Eighteen patients (40.9%) had refractory cytopenia of childhood (RCC), 19 (43.2%) had refractory anemia with excess blasts (RAEB), and 7 (15.9%) had refractory anemia with excess blasts in transformation/myelodysplastic related acute myeloid leukemia (RAEB-T/MDR-AML). Cytogenetic abnormalities were observed in 54.8% of evaluable patients, in which monosomy 7 was the commonest abnormality observed (26.1%). Allogeneic HSCT from HLA matched donors was performed in 9/44 patients. The Average CD34 + count was 3.57 × 106/Kg recipient weight. The 2-year OS and EFS for the whole cohort were 43.3% and 33.8%, respectively, and for the transplanted patients were 75% and 60%, respectively. Univariate analysis showed improved 2 years-OS and EFS in patients who underwent HSCT as compared to those who didn't (75% versus 35.4% for OS, P = 0.088 and 60% versus 27.5%, for EFS, P = 0.074). Conclusion Allografts from either matched related or unrelated or even haplo-identical donors should be offered for pediatric MDS patients, as HSCT is the only curative option for those patients. Further collaborative efforts, a more precise molecular diagnosis, as well as large-scale prospective therapeutic trials are required to develop standard diagnostic and therapeutic guidelines for such rare disease.

Outcome and factors affecting survival of childhood myelodysplastic syndrome; single centre experience

IntroductionAlthough many therapeutic options have been attempted for pediatric myelodysplastic syndrome (MDS), still hematopoietic stem cell transplant (HSCT) is the only curative therapy. We described the outcome of these patients and the factors affecting the overall survival (OS) and event free survival (EFS). MethodsThis is a retrospective descriptive study included 44 patients with MDS who presented to Children Cancer Hospital Egypt (CCHE-57357), between July 2007 and December 2015. HSCT was offered to patients who had HLA matched family donors. ResultsEighteen patients (40.9%) had refractory cytopenia of childhood (RCC), 19 (43.2%) had refractory anemia with excess blasts (RAEB), and 7 (15.9%) had refractory anemia with excess blasts in transformation/myelodysplastic related acute myeloid leukemia (RAEB-T/MDR-AML). Cytogenetic abnormalities were observed in 54.8% of evaluable patients, in which monosomy 7 was the commonest abnormality observed (26.1%). Allogeneic HSCT from HLA matched donors was performed in 9/44 patients. The Average CD34 + count was 3.57 × 106/Kg recipient weight. The 2-year OS and EFS for the whole cohort were 43.3% and 33.8%, respectively, and for the transplanted patients were 75% and 60%, respectively. Univariate analysis showed improved 2 years-OS and EFS in patients who underwent HSCT as compared to those who didn't (75% versus 35.4% for OS, P = 0.088 and 60% versus 27.5%, for EFS, P = 0.074). ConclusionAllografts from either matched related or unrelated or even haplo-identical donors should be offered for pediatric MDS patients, as HSCT is the only curative option for those patients. Further collaborative efforts, a more precise molecular diagnosis, as well as large-scale prospective therapeutic trials are required to develop standard diagnostic and therapeutic guidelines for such rare disease.

Management of Hereditary Spastic Paraplegia: A Systematic Review of the Literature.

The term hereditary spastic paraplegia (HSP) embraces a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by progressive spasticity and weakness of the lower limbs. There currently exist no specific therapies for HSP, and treatment is exclusively symptomatic, aimed at reducing muscle spasticity, and improving strength and gait. The authors set out to perform a comprehensive systematic review of the available scientific literature on the treatment of HSP, applying Cochrane Collaboration methods. The Google Scholar, PubMed and Scopus electronic databases were searched to find relevant randomized control trials (RCTs) and open-label interventional studies, prospective, and retrospective observational studies of supplements, medications, and physical therapy, as well as case reports and case series. Two authors independently analyzed 27 articles selected on the basis of a series of inclusion criteria. Applying a best-evidence synthesis approach, they evaluated these articles for methodological quality. A standardized scoring system was used to obtain interrater assessments. Disagreements were resolved by discussion. The 27 articles focused on pharmacological treatment (n = 17 articles), physical therapy (n = 5), surgical treatment (n = 5). The drugs used in the 17 articles on pharmacological therapy were: gabapentin, progabide, dalfampridine, botulinum toxin, L-Dopa, cholesterol-lowering drugs, betaine, and folinic acid. Gabapentin, progabide, dalfampridine, and botulinum toxin were used as antispastic agents; the study evaluating gabapentin efficacy was well-designed, but failed to demonstrate any significant improvement. L-Dopa, cholesterol-lowering drugs, betaine, and folinic acid were only used in specific HSP subtypes. Two of the three studies evaluating cholesterol-lowering drugs (in SPG5 patients) were well-designed and showed a significant reduction of specific serum biomarkers (oxysterols), but clinical outcomes were not evaluated. The articles focusing on physical treatment and surgical therapy were found to be of low/medium quality and, accordingly, failed to clarify the role of these approaches in HSP. Despite recent advances in understanding of the pathogenesis of HSP and the possibility, in several centers, of obtaining more precise and rapid molecular diagnoses, there is still no adequate evidence base for recommending the various published therapies. Well-designed RCTs are needed to evaluate the efficacy of both symptomatic and pathogenetic treatments.

A novel phenotype with splicing mutation identified in a Chinese family with desminopathy.

Background:Desminopathy, a hereditary myofibrillar myopathy, mainly results from the desmin gene (DES) mutations. Desminopathy involves various phenotypes, mainly including different cardiomyopathies, skeletal myopathy, and arrhythmia. Combined with genotype, it helps us precisely diagnose and treat for desminopathy.Methods:Sanger sequencing was used to characterize DES variation, and then a minigene assay was used to verify the effect of splice-site mutation on pre-mRNA splicing. Phenotypes were analyzed based on clinical characteristics associated with desminopathy.Results:A splicing mutation (c.735+1G>T) in DES was detected in the proband. A minigene assay revealed skipping of the whole exon 3 and transcription of abnormal pre-mRNA lacking 32 codons. Another affected family member who carried the identical mutation, was identified with a novel phenotype of desminopathy, non-compaction of ventricular myocardium. There were 2 different phenotypes varied in cardiomyopathy and skeletal myopathy among the 2 patients, but no significant correlation between genotype and phenotype was identified.Conclusions:We reported a novel phenotype with a splicing mutation in DES, enlarging the spectrum of phenotype in desminopathy. Molecular studies of desminopathy should promote our understanding of its pathogenesis and provide a precise molecular diagnosis of this disorder, facilitating clinical prevention and treatment at an early stage.

Next generation sequencing and imprinting disorders: Current applications and future perspectives: Lessons from Silver-Russell syndrome

Imprinting Disorders are a group of rare diseases with overlapping phenotypes which are associated with similar molecular changes and affect imprinted chromosomal regions. Clinical features mainly occur prenatally or in childhood, but have a severe lifelong impact on health. Due to their clinical and molecular heterogeneity, the diagnosis of imprinting disorders is often challenging and requires testing of a broad spectrum of genomic variants and aberrant methylation of imprinted loci (epimutations). A significant number of patients suspicious for imprinting disorders remain without a molecular confirmation, and in these cases differential diagnoses have to be considered. In fact, in patients with clinical features suggestive for imprinting disorders, the precise identification of the molecular cause is relevant for both clinical management as well as for genetic counselling. Thus, a comprehensive testing approach has to be applied. Next generation sequencing (NGS) based studies show that this technique is a valuable tool to improve the diagnostic efficiency particularly in entities with broad differential diagnoses. Furthermore, the development of diverse NGS approaches allows new insights in the function of imprinted regions, their structures, interactions and regulation. Based on a large cohort of patients referred for routine Silver Russel syndrome testing, the appropriateness and limitations of first trial tests in imprinting disorders are demonstrated in this report, but the chances of genomic NGS approaches for diagnostics and research are elucidated as well. Finally, the significance of the precise molecular diagnosis for the personalized management of the patient, and genetic counselling of the family will be discussed.

Precision medicine for acute myeloid leukemia based on genomic profiling

Recent advanced high-throughput sequencing technologies have offered insights into the molecular landscape of acute myeloid leukemia (AML), revealing its genetic heterogeneity. As recurrent alterations should be related to the molecular pathogenesis of AML, assessing the mutation profile of each patient would contribute to the precise molecular diagnosis, precise risk stratification, and appropriate treatment decisions. In fact, the most recent WHO classification and clinical guidelines for AML are categorized by genetic alterations. In addition, serial monitoring of genetic markers could be useful to detect minimal residual disease (MRD) at the time of complete remission as well as clonal changes during the disease course. Because large databases of matched clinical genomics data are needed to use complicated genomic data for clinical decision making, we have been constructing an integrated database of hematological malignancies (knonc) supported by the AMED since 2016. Precision medicine for AML based on genetic information will provide optimal target drugs and useful information for the enhancement of clinical outcome.

Technical and economic efficiency of methods for extracting genomic DNA from Meloidogyne javanica

Plant parasitic nematodes reduce the production of agricultural crops. Species diagnosis is essential to predict losses, determine economic damage levels and develop integrated pest management programs. DNA extraction techniques need to be improved for precise and rapid molecular diagnosis of nematodes. The objective of the present study was to evaluate the efficiency of DNA extraction and amplification by PCR, cost and execution time by Chelex, Worm Lysis Buffer Method (WLB), Holterman Lysis Buffer Method (HLB) and FastDNA methods for nematodes of the Meloidogyne genus. The qualitative and quantitative efficiency of DNA extraction varied between methods. The band size of the amplified PCR product with WLB, Chelex and HLB methods was 590 bp. Extraction with the FastDNA is not recommended for DNA extraction from nematodes because it results in a low DNA concentration without bands in PCR amplification, besides presenting high cost. The efficiency of the WLB method to extracting DNA from Meloidogyne javanica was greater, ensuring a higher concentration and purity of the extracted material and guaranteeing lower costs and greater ease of PCR amplification.

Targeted high-throughput sequencing technique for the molecular diagnosis of primary immunodeficiency disorders.

The aim of this study was to investigate the usefulness of targeted high-throughput sequencing (HTS) for the molecular diagnosis of primary immunodeficiency diseases (PID).A total of 56 clinically diagnosed or suspected PID patients were divided into 4 groups according to the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015 and their chief clinical presentations. Patients and their biological family members were examined by targeted HTS, which sequenced the exons and ±10 bp flanking introns of 171 PID-related genes panel. All significant variants were confirmed by PCR-Sanger sequencing. Pathogenicity of the variants was evaluated by using bioinformatics.A total of 117 variants in 73 genes were found in 56 patients. Accurate molecular diagnosis of PID was made in 13 (23.2%) patients, and 12 novel mutations were detected in these patients. Twenty-seven patients carried heterozygous variants that are probably pathogenic in ≥2 genes; 16 patients had only 1 missense variant, or had several variants but not >1 variant was deleterious as evaluated by bioinformatics. The meaning of the targeted HTS results of these patients remains to be studied.Targeted HTS can make a precise molecular diagnosis of PID and detect more novel pathogenic mutations. More and more variations with ambiguous significance are discovered and explanation of these variations is a challenge to the clinicians.