Molecular Analysis Of Mutations Research Articles

Complex formation and reciprocal regulation between GSK3β and C3G

GSK3β, a ubiquitously expressed Ser/Thr kinase, regulates cell metabolism, proliferation and differentiation. Its activity is spatially and temporally regulated dependent on external stimuli and interacting partners, and its deregulation is associated with various human disorders. In this study, we identify C3G (RapGEF1), a protein essential for mammalian embryonic development as an interacting partner and substrate of GSK3β. In vivo and in vitro interaction assays demonstrated that GSK3β and Akt are present in complex with C3G. Molecular modelling and mutational analysis identified a domain in C3G that aids interaction with GSK3β, and overlaps with its nuclear export sequence. GSK3β phosphorylates C3G on primed as well as unprimed sites, and regulates its subcellular localization. Over-expression of C3G resulted in activation of Akt and inactivation of GSK3β. Huntingtin aggregate formation, dependent on GSK3β inhibition, was enhanced upon C3G overexpression. Stable clones of C2C12 cells generated by CRISPR/Cas9 mediated knockdown of C3G, that cannot differentiate, show reduced Akt activity and S9-GSK3β phosphorylation compared to wild type cells. Co-expression of catalytically active GSK3β inhibited C3G induced myocyte differentiation. C3G mutant defective for GSK3β phosphorylation, does not alter S9-GSK3β phosphorylation and, is compromised for inducing myocyte differentiation. Our results show complex formation and reciprocal regulation between GSK3β and C3G. We have identified a novel function of C3G as a negative regulator of GSK3β, a property important for its ability to induce myogenic differentiation.

Case report: Pitt-Hopkins like syndrome with CNTNAP2 mutation

BackgroundPitt-Hopkins syndrome (PHS) is a rare cause of severe intellectual disability, seizures, language impairment, and peculiar facial dysmorphism. It is caused by a mutation in transcription factor 4 (TCF4). Through molecular karyotyping and mutational analysis, a study identified recessive defects in two genes, contactin associated protein like 2 (CNTNAP2) and Neurexin I (NRXN1), in patients with similar presentations of Pitt-Hopkins syndrome and called Pitt-Hopkins-like syndrome (Zweier et al., J Med Genet 80: 994-1001, 2007).We present the first case report of a child in Iraq with Pitt-Hopkins-like syndrome that was referred to the Welfare Children’s Hospital/Medical City of Baghdad because of her intellectual disability.Case presentationThe patient was 4-year-old female child who presented with psychomotor delay and language impairment. She had frequent attacks of the respiratory tract and eye infections. Ophthalmologic examination revealed left-sided esotropia and severe myopia. Routine hematologic, serologic, and chemistry tests were within normal ranges. EEG revealed diffuse theta slowing and diffuse beta activity. The audiological test was normal. NCS and EMG showed normal results. Echo study, chest X-ray, and abdominal/pelvic ultrasound revealed normal findings. Brain MRI showed mild bilateral frontal-temporal atrophy. Whole-exome sequencing (WES) revealed a homozygous stop mutation in CNTNAP2 with a heterozygous state in both parents.ConclusionIntellectual disability may result from different types of abnormal cellular processes and with widening the use of molecular gene analysis in cases of intellectual disability, underdiagnosed cases of Pitt-Hopkins and Pitt-Hopkins-like syndromes may be uncovered.

Biochemical and Structural Investigation of GnnA in the Lipopolysaccharide Biosynthesis Pathway of Acidithiobacillus ferrooxidans.

Lipopolysaccharide (LPS) is a crucial component in the outer membrane of Gram-negative bacteria that contributes to both pathogenicity as well as immunity against pathogenic bacteria. Typical LPS contains GlcN disaccharide as the core of lipid A. However, some bacteria such as Acidithiobacillus ferrooxidans and Leptospira interrogans contain GlcN3N in lipid A instead. This modification has been shown to dampen the host immune response and increase resistance to antimicrobial peptides. Therefore, investigation of the enzymes responsible for the biosynthesis of GlcN3N has promising applications in the development of vaccines, antibiotics, or usage of the enzymes in chemoenzymatic synthesis of modified LPS. Here, we describe biochemical and structural investigation of GnnA from A. ferrooxidans (AfGnnA) that is responsible for oxidation of UDP-GlcNAc, which subsequently undergoes transamination to produce UDP-GlcNAc3N as a precursor for LPS biosynthesis. AfGnnA is specific for NAD+ and UDP-GlcNAc. The crystal structures of AfGnnA in combination with molecular dynamics simulation and mutational analysis suggest the substrate recognition mode and the catalytic mechanism. K91 or H164 is a potential catalytic base in the oxidation reaction. The results will not only provide insights into the biosynthesis of unusual LPS but will also lay the foundation for development of more immunogenic vaccines, novel antibiotics, or utilization of GnnA in the synthesis of UDP-sugars or modified LPS.

Bi-allelic mutations in EGR2 cause autosomal recessive demyelinating neuropathy by disrupting the EGR2-NAB complex.

Mutations in the early growth response 2 gene (EGR2) cause demyelinating, but also axonal, neuropathies differing in severity and age of onset. Except for one family, all reported cases have autosomal dominant inheritance and mutations are localized within the three zinc finger (ZNF) DNA-binding domain. The aim of this study was to provide a clinical and molecular analysis of a novel recessive mutation in EGR2. Clinical and electrophysiological assessments of three affected patients, from a consanguineous family, were performed. Genetic analyses of EGR2 were carried out by Sanger sequencing. Functional effects of clinical recessive mutations were assessed using a mammalian two-hybrid assay. A novel missense mutation (c.791C>T; p.P264L) in the homozygous state was detected outside the ZNF domains of the EGR2 gene. Three affected siblings presented with distal demyelinating polyneuropathy with severe sensory loss, progressive thoracolumbar scoliosis and trigeminal neuralgia. Respiratory compromise and cranial nerve dysfunction were also found. Our data indicate that the p.P264L mutation prevents interaction of EGR2 transcription factor with NAB corepressors, suggesting that a disruption of the NAB-EGR2 protein interactions can result in dramatic neuropathy. Mutations in, or next to, the R1 domain of EGR2 should be considered with extreme caution for genetic counseling, since these could cause a severe neuropathy with an autosomal recessive manner of transmission.

Factor VIII Intron 22 Inversion in Severe Hemophilia A Patients in Palestine.

Background Hemophilia A is an X-linked recessive bleeding disorder caused by mutations in FVIII gene with an incidence of 1 in 5,000 to 10,000 live born males. The Inv22 mutation is a major cause of the disease worldwide, accounting for up to 40%–50% of severe FVIII mutations. The aim of the present study was to screen Inv22 of the FVIII gene in Palestinian patients with severe HA and reveal its role as a predisposing factor for the development of inhibitors. Materials and Methods A cohort of 77 HA individuals including 5 carrier females from 52 unrelated families registered at governmental hemophilia centers in the West Bank area of Palestine was investigated. The demographic data and the clinical history were retrieved from medical files. Molecular analysis of Inv22 mutation in severe HA (30 cases) from Palestine was performed using the subcycling polymerase reaction (S-PCR). FVIII coagulant activities were carried out on an aPTT-based 1-stage clotting assay. FVIII inhibitors were quantified using the Nijmegen modification of the Bethesda assay. Result Overall, 41.7% (30/72) of the studied cases were classified as having severe HA, 22.2% (16/72) had moderate HA, and 36.1% (26/72) had mild HA. Five randomly selected carrier mothers were screened for the Inv22 mutation to confirm its transmission to their sons. The Inv22 mutation was detected in 11 severe HA patients (36.6%). Among the severe HA patients with positive Inv22, 45.5% (5/11) had developed inhibitors. The current study showed that there was no association (p=0.53) between inhibitor development and the Inv22 mutation. Conclusion Findings on Inv22 are in agreement with worldwide reports, being a major genetic mutation in severe HA. The S-PCR is a simple, rapid, and cost-effective method for the diagnosis of Inv22 in severe HA patients. Although the Inv22 mutation was associated with 36.6% of severe HA phenotype cases, it was not a major predisposing factor for inhibitor formation.

Molecular Analysis of Oncogenic Mutations in Resected Margins by Next-Generation Sequencing Predicts Relapse in Non-Small Cell Lung Cancer Patients.

ObjectiveTo investigate the genetic mutations in both tumor and marginal tissues in patients with non-small cell lung cancer (NSCLC), and to evaluate the potential prognostic value in patients with margins gene positive.MethodsNext-generation sequencing (NGS) technique was used to detect genetic mutation in tumor and marginal tissues of the bronchus in 88 patients with NSCLC. Correlation of genetic mutations with pathology, lymph node metastasis, disease-free survival and overall survival was analyzed.ResultsOf the 88 patients, 83 cases (94.3%) had gene mutations in the tumor samples and 12 cases (13.6%) had genetic alterations in their margins. Most of the gene mutations detected were cancer drivers. Six common driver genes between tumor and marginal tissues were identified, including EGFR, TP53, CDKN2A, CTNNB1, BRAF, and NF1. Kaplan–Meier analysis revealed that the median disease-free survival (DFS) was significantly shorter in patients with detectable gene mutations in marginal tissues compared with patients without mutations in margins (30.7 versus 24.4 months, log-rank χ2 = 4.78, P =0.029). Consistently, a shorter median OS was observed in patients harboring gene mutations in margins compared with patients with no mutations in margins (49.1 versus 32.2 months, log-rank χ2 = 3.669, P =0.055).ConclusionThese findings identify the presence of oncogenic alterations in microscopically negative margins in NSCLC patients associated with elevated risk of relapse and shorter survival time. Thus, examination of microscopically negative margins by NGS represents a valuable approach to predict the clinical outcome of NSCLC patients.

Evolving sequence mutations in the Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

BackgroundMiddle East respiratory syndrome coronavirus (MERS-CoV) has continued to cause sporadic outbreaks of severe respiratory tract infection over the last 8 years. MethodsComplete genome sequencing using next-generation sequencing was performed for MERS-CoV isolates from cases that occurred in Riyadh between 2015 and 2019. Phylogenetic analysis and molecular mutational analysis were carried out to investigate disease severity. ResultsA total of eight MERS-CoV isolates were subjected to complete genome sequencing. Phylogenetic analysis resulted in the assembly of 7/8 sequences within lineage 3 and one sequence within lineage 4 showing complex genomic recombination. The isolates contained a variety of unique amino acid substitutions in ORF1ab (41), the N protein (10), the S protein (9) and ORF4b (5). ConclusionOur study shows that MERS-CoV is evolving. The emergence of new variants carries the potential for increased virulence and could impose a challenge to the global health system. We recommend the sequencing every new MERS-CoV isolate to observe the changes in the virus and relate them to clinical outcomes.

High frequency of BRCA recurrent mutations in a consecutive series of unselected ovarian cancer patients

Abstract Hereditary predisposition to breast and ovarian cancer (HBOC) is diagnosed by molecular analysis of deleterious mutations in BRCA genes, allowing oncogenetic follow-up of patients and of their families. BRCA testing addresses only to HBOC families, using restrictive inclusion criteria based on familial history of cancer and age at diagnosis. Sporadic ovarian cancer has high incidence and mortality in Romania, with low median age of diagnosis and possibly a higher magnitude of hereditary contribution comparing to othe populations. However, sporadic ovarian cancers do not qualify for BRCA testing according to inclusion criteria, and a complete BRCA screening of all cancers is neither feasible nor recommended. Despite the large diversity of BRCA mutations worldwide, some recurrent mutations have higher frequencies in diverse populations. Precisely screening for recurrent mutations in a target population allows to rapidly identifying mutation carriers without sequencing the entire BRCA genes. In Romanian population and neighboring countries, several recurrent mutations have already been described. In a consecutive series of 50 sporadic ovarian cancer patients, not qualifying for BRCA complete testing, we screened for 9 most common BRCA mutations, by multiplex-PCR, RFLP and targeted Sanger sequencing. Our results revealed 6 different BRCA mutations in 8 unrelated patients, with a frequency of 16%, much higher than expected. We further recommend screening for the identified mutations in larger series of cancer patients. The results are highly beneficial to cancer patients, healthy relatives, and overall, considering prevention in cancer a priority, to public health system and future of oncogenetics in Romania

Molecular Analysis of the ABCA4 Gene Mutations in Patients with Stargardt Disease Using Human Hair Follicles

ABCA4 gene mutations are the cause of a spectrum of ABCA4 retinopathies, and the most common juvenile macular degeneration is called Stargardt disease. ABCA4 has previously been observed almost exclusively in the retina. Therefore, studying the functional consequences of ABCA4 variants has required advanced molecular analysis techniques. The aim of the present study was to evaluate whether human hair follicles may be used for molecular analysis of the ABCA4 gene splice-site variants in patients with ABCA4 retinopathies. We assessed ABCA4 expression in hair follicles and skin at mRNA and protein levels by means of real-time PCR and Western blot analyses, respectively. We performed cDNA sequencing to reveal the presence of full-length ABCA4 transcripts and analyzed ABCA4 transcripts from three patients with Stargardt disease carrying different splice-site ABCA4 variants: c.5312+1G>A, c.5312+2T>G and c.5836-3C>A. cDNA analysis revealed that c.5312+1G>A, c.5312+2T>G variants led to the skipping of exon 37, and the c.5836-3C>A variant resulted in the insertion of 30 nucleotides into the transcript. Our results strongly argue for the use of hair follicles as a model for the molecular analysis of the pathogenicity of ABCA4 variants in patients with ABCA4 retinopathies.

A case of oral metastasis of rectal adenocarcinoma with KRAS mutation in patient with synchronous lung adenocarcinoma

AbstractDetecting the origin of the primary cancer that metastasizes in the oral cavity in patients with multiple primary cancers is challenging because of its rarity and complexity. We present an unusual case of an 86‐year‐old women with a maxillary gingival tumor that was histologically proven to be adenocarcinoma; she had been diagnosed with rectal and lung adenocarcinoma. A multidisciplinary approach involving complementary molecular analysis of KRAS gene mutation helped in the diagnosis of metastatic gingival tumor from rectal cancer.

MODERN PROBLEMS OF АLPORT SYNDROME DIAGNOSIS

Introduction. In the practice of adult nephrologists there are cases of rare genetically caused kidney damage, in particular, Alport syndrome. This is due to the availability and improvement of instrumental diagnostic methods, timely approaches to treatment in pediatric practice, and prolongation of the pre-dialysis period. 
 Goal. Analysis and synthesis of new data from domestic and foreign sources on the etiology, pathogenesis, clinical manifestations, types of inheritance, differential diagnosis of Alport syndrome in order to improve the success of students, interns and teachers in the study of nephrological subjects. 
 Material and methods. Review of contemporary and foreign literary sources; techniques – description, analysis, abstracting. 
 Results. Alport syndrome (AS, synonym: hereditary nephritis) is non-immune genetically determined glomerulopathy caused by a mutation of genes that encode collagen type IV of basement membranes, manifested by hematuria and / or proteinuria, a progressive decreased renal function, combined with pathology of hearing and abnormalities affecting the eyes. Alport syndrome inherited type: X-linked dominant (XLAS): 85%, autosomal recessive (ARAS): 15%, autosomal dominant (ADAS): 1%. 
 Conclusions. Family history, electron microscopy, immunochemical analysis of type IV collagen expression are informative for verifying the diagnosis of Alport syndrome. Due to the rarity of this disease, in addition, the fact that patients often refuse kidney biopsy, it is necessary to cooperate more closely with genetic laboratories, to take measures to improve the availability of molecular analysis of mutations of collagen IV genes. In addition, it is a fact that sometimes the family history of the disease is ambiguous, unavailable for genetic analysis, and patients refuse to have a kidney biopsy. It is a motive to encourage doctors to improve their educational work with patients about safety of this analysis and its value.

Molecular Analysis of Mutations in the Human HPRT Gene.

The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and the products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.

Wilson’s Disease in Children : An Update

Wilson’s Disease is an autosomal recessive disorder of copper metabolism due to ATP7B gene defect. This defect result in progressive toxic accumulation of copper in liver, CNS, cornea, skeletal system and other organs. Clinical presentations of Wilson’s disease (WD) in childhood ranges from asymptomatic liver disease to cirrhosis or acute liver failure, whereas neurological and psychiatric symptoms are rare. The basic diagnostic approach includes serum ceruloplasmin and 24-hour urinary copper excretion. Final diagnosis of WD can be established using a diagnostic scoring system based on symptoms, biochemical tests assessing copper metabolism, and molecular analysis of mutations in the ATP7B gene. Pharmacological treatment is life-long and aims at removal of copper excess by chelating agents as D-penicillamine, trientine or inhibition of intestinal copper absorption with zinc salts. Acute liver failure often requires liver transplantation. Genetic therapy and haplocyte transplantation represent future curative treatment for Wilson’s disease.

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

Molecular Study of FLT3 gene mutations in Acute Myeloid Leukemia from Pakistan : Correlation with clinicopathological parameters.

Introduction: FLT3 mutations are common genetic changes reported to have prognostic significance in acute myeloid leukemia (AML). Bone marrow/peripheral blood samples of 63 AML Pakistani patients were collected and DNA was isolated.Materials and Methods: The FLT3 internal tandem duplication (ITD) and the D835 activating mutation in the tyrosine kinase domain (TKD) were analyzed by polymerase chain reaction (PCR).Results: Among 63 AML patients, 42 were males and 21 were females with male to female ratio 2.1:1. The age ranged between 15 to 75 years with a median age of 32 years. AML-M2 was the predominant French-American-British (FAB) subtype (32%) followed by M3 (27%), M4 (19%), M5 (6.3%) and M1 (6.3%). The incidence of FLT3/ITD and TKD was 22% and 6.3% respectively. Majority of the FLT3/ITD mutation were detected in AML-M4 (38%) patients while D835 mutation was common in both FAB M1, M2. Presence of mutation was significantly associated with age but significance was not achieved for hyperleukocytosis.Conclusion: This study constitutes the first report from Pakistan reporting significant presence of FLT3/ITD mutations in our adult AML patients with different FAB subtypes Molecular mutation analysis in different cytogenetic groups with follow-up is required to understand the pathogenesis of leukemias and their role as a valuable prognostic marker in our patients.

In-silico molecular characterization and mutational analysis of inter-alpha-trypsin inhibitor heavy chain 4 in rheumatoid arthritis

Inter-alpha-trypsin inhibitor heavy chain (ITIH4) is IL-6-dependent acute phase protein. Increased expression of ITIH4 during acute phase inflammation of Rheumatoid Arthritis (RA) was reported earlier. Analyzing the heavy chain would, therefore, be crucial to understand the regulation and localisation of Serum-derived Hyaluronic acid-Associated Protein (SHAP) complex in acute phase inflamed conditions. In our previous studies, we have computationally constructed the 3D model of ITIH4 and checked its relevance. But further detailed structural annotation of ITIH4 is required to reveal its surface properties, domain characterisation, and molecular level interaction including drug designing. In the present study, we have utilized our constructed high-resolution three-dimensional (3D) model of ITIH4 protein to reveal the associated surface parameters and its functional role based on the structure-based homology search. Signal peptide prediction has been carried out by the Signalp 4.1 server, whereas COFACTOR server has been utilized for the prediction of active site residues, ligand binding residues, and gene ontology and enzyme classification. Immune epitope database (IEDB) analysis resource was used for the identification of antigenic peptides and Disease-Susceptibility-based SAV Phenotype Prediction (SUSPECT) server revealed the mutation prone residues of ITIH4. Present study indicated that mutation L304H and L304P may enable the protein model highly unstable. The molecular dynamic simulation was carried out for a comparative analysis between native and mutant protein. Additionally, interaction studies implicate that ITIH4 may modulate T cell-mediated inflammation in synovial region during RA. In-silico construction of ITIH4 mutants with single amino acid change may prove to be a tool in experimental studies to understand the importance of each functional domain.

Germline and somatic mutations in patients with multiple primary melanomas: a next generation sequencing study

IntroductionMultiple primary melanomas (MPM) occur up to 8% of patients with cutaneous malignant melanoma (CMM). They are often sporadic harbouring several somatic mutations, but also familial cases harbouring a CDKN2A germline mutation have been describe in Caucasian populations. The aim of this study was to investigate the incidence, the distribution patterns and the impact of known and unknown germline and somatic mutations in patients with MPM from Italy.Materials and methodsOne-hundred and two MPM patients were enrolled for germline mutation analysis, and five patients with at least four MPMs were identified for somatic mutation analysis. The demographic, pathologic and clinical features were retrieved from medical records. Molecular analysis for both germline and somatic mutations was performed in genomic DNA from peripheral blood and tissue samples, respectively, through a next generation sequencing approach, using a specific multiple-gene panel constructed by the Italian Melanoma Intergroup for somatic analysis and a commercial cancer hotspot panel for somatic analysis.ResultsCDKN2A mutations were detected in 6/16 (37.5%) and 3/86 (3.5%) MPM cases with and without family history for melanoma, respectively. Furthermore, multiple MC1R and, to a lesser extent, ATM variants have been identified. BAP1 variants were found only in MPM patients from southern Italy. The most frequent somatic variants were the pathogenic BRAFV600E and TP53, followed by KIT, PIK3CA, KDR, and NRAS. Single APC, ERBB4, MET, JAK3 and other variants with unknown function were also detected.ConclusionsCDNK2A mutation is the most relevant susceptibility mutation in Italian patients with MPM, especially those with a family history for CMM. The prevalence of this mutation and other sequence variants identified in this study varies among specific sub-populations. Furthermore, some heterogeneity in driver somatic mutations between sporadic MPMs has been observed, as well as in a number of associated sequence variants the clinical impact of which needs to be further elucidated.

Molecular analysis of V617F mutation in Janus kinase 2 gene of breast cancer patients.

BackgroundBreast cancer is a multifactorial disease with the highest frequency in females. Genetic and environmental factors can cause mutation in several genes like tyrosine kinase, JAK2 gene which may initiate cancer. Molecular analysis of mutations in the JAK2 gene along with determination of environmental, clinical and haematological risk factors associated with breast cancer patients is need of hour to improve patient's healthcare. Somatic JAK2 valine-to-phenylalanine (617 codon) mutation is one of the widely prevalent mutations.MethodsBlood was collected from seventy breast cancer patients after their consent. The questionnaire included risk factors, age group, locality, number of children, tumor type, family history, time of initial diagnosis, no of cycles/month, water conditions and exposure to radiations. Molecular analysis were carried out from genomic DNA using Sanger sequencing and allele-specific PCR to check the V617F point mutation.ResultsThe breast cancer risk factors includes unfiltered water (68.57%), urban (58.57%), menopause (55.71%), family history of cancer (18.57%), tumor grades (II, 37.14% and III, 35.71%), consanguineous marriages (44.28%) and having more than 3–4 children (45.71%). Prevalence of breast cancer was higher after the age of 35 and maximum at 35–50. In allele-specific PCR of 70 patients, 25 patients were wild type (229 bp), 25 patients were with partially deleted gene (200 bp), and 20 patient had shown no or less than 40 bp size fragments. In Sanger's sequencing of 70 BC cases, 18% were found to be positive for V617F point mutation, including 6 homozygous (T/T) and 7 heterozygous (G/T) mutations at nucleotide position 1849 in exon 14 of the JAK2 gene.ConclusionsEnvironmental and clinical risk factors were associated with breast cancer which can be overcome by improving awareness of associated risks, health facilities and reducing stress.

62. SUCCESSFUL STRATEGY OF COMPREHENSIVE PRE-IMPLANTATION GENETIC TESTING FOR DUCHENNE MUSCULAR DYSTROPHY AND CHROMOSOME BALANCE USING KARYOMAPPING

Introduction Duchenne muscular dystrophy (DMD) is one of the most common muscular dystrophy in childhood. The patients suffer progressive proximal muscle weakness and die from respiratory failure and cardiomyopathy in their 30’s. Pre-implantation genetic testing (PGT) is an alternative traditional prenatal diagnosis (PND). However, extremely large size of the gene and the diversity of mutations cause molecular genetic testing difficult and labor intensive. Modern haplotyping using aSNP and Karyomapping algorithm would be useful for diagnosis of monogenic disease. This study applied Karyomapping for PGT-M of DMD in 2 clinical PGT cycles in comparison to standard PCR testing techniques. Material and methods Two families at risk of having DMD offspring joined the project following thoroughly counselling and inform consent was obtained. The patients underwent IVF procedures. Embryo biopsy was performed on Day-5 post-fertilization and biopsied trophectoderm underwent whole genome amplification. SNP array with Karyomapping analysis was carried out for haplotyping as well as copy number variation (CNV). Multiplex PCR with mini-sequencing was performed alongside for confirmation standard molecular mutation analysis. Multiple microsatellites within dystrophin gene were also analyzed for linkage analysis and contamination identification. Results Nine embryos with good morphology from each patient were chosen for PGT. Karyomapping results of family TZ (DMD c.895G>T) revealed three normal, two carriers, two affected and two with intragenic recombination. Standard mutation analysis using multiplex fluorescent PCR incorporation with mini-sequencing and microsatellites analysis for linkage analysis confirmed haplotyping results in all embryos. In addition, karyomapping demonstrated one embryo with chromosome unbalanced, i.e. 46,XX, -6q (intragenic recombination). Therefore, three normal (two male and one female) and two carrier (both female) embryos with chromosomally balanced were fulfilled for transfer. During the first embryo transfer, one normal female embryo was chosen, no pregnancy was resulted. In the second transfer, one normal male embryo was transferred, one ongoing pregnancy was resulted. Karyomapping results of family JM (DMD exon 8-9 duplication) revealed two normal, two carriers, two affected and one with intragenic recombination. Standard microsatellites analysis for linkage analysis confirmed haplotyping results in all embryos. Additionally, karyomapping demonstrated one embryo with chromosome unbalanced, i.e. 45,XX, +2P,-22 (normal) and one embryo with uniparental disomy (UPD), i.e. 46,XX (normal). Therefore, two normal (both female) and two carrier (both female) embryos with chromosomally balanced embryos were fulfilled for transfer. All are being frozen waiting for transfer. Polymorphic marker analysis revealed the absence of extraneous DNA contamination. Conclusions Two clinical PGT-M cycles using karyomapping were performed for two families at risk of having DMD offspring. This study exhibits that aSNP provides the benefit of extra information of chromosome balance and parental origins, i.e. uniparental disomy in one of the embryo. Therefore, kryomapping can omit the risk of transfer chromosomally unbalanced embryos, termination of abnormal chromosome pregnancy later and the birth of abnormal chromosome babies. Therefore, karyomapping provides an accurate, quick, time saving for protocol development, universal PGT-M method for every monogenic disease of various types of mutations and also the advantage of CNV and parental origin information which is common abnormalities in pre-implantation embryos.

65. SUCCESSFUL STRATEGY OF COMPREHENSIVE PRE-IMPLANTATION GENETIC TESTING FOR BETA-THALASSEMIA-HEMOGLOBIN E DISEASE AND CHROMOSOME BALANCE USING KARYOMAPPING

Introduction Thalassemia syndrome and hemoglobinopathy are the world commonest monogenic disease and cause health and economic burden worldwide. Pre-implantation genetic testing (PGT) is an alternative to traditional prenatal diagnosis (PND). However, the nature of a vast variety of mutations makes molecular genetic testing sophisticated and labor intensive. Modern haplotyping using SNP array (aSNP) and Karyomapping algorithm would omit molecular analysis development process and provide chromosome balance information at the same time. This study applied Karyomapping for PGT-M of beta-thalassemia-hemoglobin E (Hb E) disease in 2 clinical PGT cycles in comparison to PCR testing techniques. Material and methods: Two families at risk of having beta-thalassemia-Hb E disesase offspring decided to join the project following thoroughly counselling and inform consent was obtained. The patients underwent routine IVF procedures. Embryo biopsy was performed on Day-5 post-fertilization and the biopsied trophectoderm underwent whole genome amplification. SNP array with Karyomapping analysis was carried out for haplotyping as well as copy number variation (CNV). Multiplex PCR with mini-sequencing was performed alongside for confirmation standard molecular mutation analysis. Informative polymorphic marker was also included for contamination identification Results Thirteen and nine embryos with good morphology from families YW and CS were chosen for PGT, respectively. Karyomapping results of family YW (beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease) revealed four normal, two beta-thalassemia trait, one Hb E trait and six affected with beta-thalassemia-Hb E disease. Standard mutation analysis using multiplex fluorescent PCR and mini-sequencing confirmed haplotyping results in all embryos. In addition, Karyomapping demonstrated three embryos with chromosome unbalanced, i.e. 45,XY, -19 (affected), 47,XY, +16 (beta-thalassemia trait) and 47,XX, +21 (beta-thalassemia trait). Therefore, four normal (three male and one female) and one Hb E trait embryos were fulfilled for transfer. One normal male embryo was chosen for transfer and one normal male baby was delivered. Prenatal and postnatal DNA testing confirmed PGT results. Karyomapping results of family CS (beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease) revealed six Hb E trait and three affected with beta-thalassemia-Hb E disease. Standard mutation analysis using multiplex fluorescent PCR and mini-sequencing confirmed haplotyping results in all embryos. Additionally, Karyomapping demonstrated two embryos with chromosome unbalanced, i.e. 45,XY, -21 (affected) and 45,XY, -22 (Hb E trait). Therefore, five Hb E trait embryos were fulfilled for transfer. One Hb E trait embryo was chosen for transfer and one ongoing pregnancy was resulted. Polymorphic marker analysis revealed the absence of extraneous DNA contamination. Conclusions Two clinical PGT-M cycles using Karyomapping were performed for two families at risk of having beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease offspring. Additional standard multiplex PCR with mini-sequencing analysis confirmed haplotyping results of Karyomapping. However, aSNP provides the benefit of extra information of chromosome balance. This study demonstrated that kryomapping can omit the risk of transfer chromosomally unbalanced embryos, termination of abnormal chromosome pregnancy later and the birth of abnormal chromosome babies. Therefore, Karyomapping provides an accurate, quick, time saving for protocol development, universal PGT-M method for every monogenic disease, and also the advantage of CNV information which is common in pre-implantation embryos.