scholarly journals Gene mapping in an anophthalmic pedigree of a consanguineous Pakistani family opened new horizons for research

2016 ◽  
Vol 19 (1) ◽  
pp. 77-84
Author(s):  
S Saleha ◽  
M Ajmal ◽  
S Zafar ◽  
A Hameed
Keyword(s):  
Linkage Mapping ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Ocular Tissue ◽  
Inherited Disease ◽  
Pakistani Family ◽  
New Horizons ◽  
Family Pedigree ◽  
Environmental Causes ◽  
Short Tandem

ABSTRACTClinical anophthalmia is a rare inherited disease of the eye and phenotype refers to the absence of ocular tissue in the orbit of eye. Patients may have unilateral or bilateral anophthalmia, and generally have short palpebral fissures and small orbits. Anophthalmia may be isolated or associated with a broader syndrome and may have genetic or environmental causes. However, genetic cause has been defined in only a small proportion of cases, therefore, a consanguineous Pakistani family of the Pashtoon ethnic group, with isolated clinical anophthalmia was investigated using linkage mapping. A family pedigree was created to trace the possible mode of inheritance of the disease. Blood samples were collected from affected as well as normal members of this family, and screened for disease-associated mutations. This family was analyzed for linkage to all the known loci of clinical anophthalmia, using microsatellite short tandem repeat (STR) markers. Direct sequencing was performed to find out disease-associated mutations in the candidate gene. This family with isolated clinical anophthalmia, was mapped to the SOX2 gene that is located at chromosome 3q26.3-q27. However, on exonic and regulatory regions mutation screening of the SOX2 gene, the disease-associated mutation was not identified. It showed that another gene responsible for development of the eye might be present at chromosome 3q26.3-q27 and needs to be identified and screened for the disease-associated mutation in this family.

scholarly journals A novel detection method for heteroduplex DNA using carbodiimide-induced interrupted primer extension

2010 ◽  
Vol 1 (1) ◽  
pp. 5
Author(s):  
Tania Tabone ◽  
Richard Cotton ◽  
Ninan Mathew ◽  
J. Des Parkin ◽  
Judy Savige
Keyword(s):  
Novel Mutation ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Screening Method ◽  
Primer Extension ◽  
Cystic Kidney Disease ◽  
Primer Binding Site ◽  
Cystic Kidney ◽  
Renal Diseases ◽  
Inherited Disease

Direct sequencing may be problematic in demonstrating mutations where inherited disease results from multiple different heterozygous variants in large genes. We describe here a novel mutation screening method based on the ability of carbodiimide to bind mismatched DNA and interrupt primer extension thereby identifying both a heterozygous variant and its location. This assay detected all four classes of DNA mismatch in 550 bp engineered plasmid fragments and in two dominantly inherited renal diseases. In patients with thin basement membrane nephropathy, the method demonstrated multiple variants within a single amplicon including some close to the primer binding site. This method also detected a complex mutation in medullary cystic kidney disease type 2 (c.278-289 del/insCCGGCTCCT) as multiple termination events and, furthermore, correctly identified five affected and 28 unaffected family members. Carbodiimide-induced interrupted primer extension identifies heterozygous variants in large or multiexonic genes, where the variants differ in each family, their locations are unknown, and even if there are multiple known non-pathogenic variants within the same amplicon. This assay incorporates a “universal” protocol that detects all types of mutations without the need for further optimization, and potentially detects mutations where the proportion of heteroduplex is less than 50%.

A Novel Denaturing-High Performance Liquid Chromatography (D-HPLC)-Based Method for Kit Mutation Screening of Patients (pts) with Systemic Mastocytosis (SM) Allows the Identification of Unreported Kit Variants.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4849-4849
Author(s):  
Sabrina Colarossi ◽  
Simona Soverini ◽  
Alessandra Gnani ◽  
Michela Rondoni ◽  
Simona Gatto ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Point Mutation ◽  
Hplc Analysis ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Rflp Analysis ◽  
Rt Pcr ◽  
Kit Mutation ◽  
Pcr Product ◽  
Tm Domain

Abstract SM is characterized by activating mutations of the Kit tyrosine kinase receptor. While the so-called ‘enzimatic site’ (ES) type mutation D816V renders Kit resistant to imatinib, ‘regulatory’ type mutations are sensitive to inhibition. Kit mutation screening with sensitive methods is important for appropriate therapeutic management of SM. Our aims were: to set up and optimize a D-HPLC-based screening method for mutations in different critical regions of the kit receptor; to assess the sensitivity and reliability of our D-HPLC assay as compared to RFLP analysis; to characterize additional mutations/polymorphisms. The analysis was performed on 37 SM pts. For each sample, a RT-PCR product spanning the catalytic and activation loops in the ES was screened in parallel by D-HPLC, followed by sequencing of D-HPLC-positive cases, and by RFLP according to an already reported method for the detection of the D816V. By RFLP analysis, 24/37 (65%) pts were positive for the D816V. By D-HPLC analysis, an abnormal eluition profile was seen in 26/37 (70%) pts - all the 24 pts scored as mutated by RFLP as well as two additional pts. Direct sequencing confirmed the presence of the D816V in all the 24 RFLP-positive cases, and showed that in two of these cases a I798I polymorphism was also present. The two pts scored positive by D-HPLC but negative by RFLP were found to have the same I798I polymorphism. The 11 pts who did not harbour ES type mutations were further investigated by D-HPLC analysis of a RT-PCR product corresponding to the transmembrane (TM) and juxtamembrane (JM) domains. D-HPLC showed an abnormal elution profile in 4 pts. Direct sequencing confirmed the presence of a point mutation in all cases. One patient showed a silent mutation at codon 546. Three pts showed a novel point mutation at codon 541 in the TM domain, resulting in a Met to Leu amino acid substitution. This is the second Kit TM domain mutation reported in a human disease and further supports the hypothesis of a role of the TM domain in regulating the enzymatic activity of an otherwise normal catalytic site. Further characterization of this novel mutant is ongoing. Morphologic and cytofluorimetric analyses of bone marrow biopsies and aspirates will be compared in order to assess whether the pts share any peculiar pathologic feature. Cos-7 cells are currently being transfected with M541L, D816V and wild-type kit in order to evaluate the effects of the M541L on kit enzymatic activity by western blot analysis of total and phosphorylated kit. Patient mast cells will be cultured in the presence or absence of kit ligand or imatinib, dasatinib and nilotinib in order to assess the sensitivity of the M541L to different kit inhibitors. Our novel D-HPLC-based assay proved a straightforward, reliable and sensitive method for kit mutation analysis. It also highlighted the importance of screening for mutations other than the D816V. D-HPLC analysis allowed us to find a novel M541L mutation which is now under characterization.

Compound heterozygous CNGA3 mutations (R436W, L633P) in a Japanese patient with congenital achromatopsia

2006 ◽  
Vol 23 (3-4) ◽  
pp. 395-402 ◽  
Author(s):  
SATOSHI GOTO-OMOTO ◽  
TAKAAKI HAYASHI ◽  
TAMAKI GEKKA ◽  
AKIKO KUBO ◽  
TOMOKAZU TAKEUCHI ◽  
...  
Keyword(s):  
Visual Acuity ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Venous Blood ◽  
Japanese Patients ◽  
White Background ◽  
Compound Heterozygous ◽  
Polymorphism Analysis ◽  
Missense Mutations ◽  
Α Subunit

Congenital achromatopsia is a stationary retinal disorder with autosomal recessive inheritance that is characterized by loss of color discrimination, low visual acuity, photophobia, and nystagmus. This disorder has been shown to be associated with CNGA3, CNGB3, and GNAT2 mutations, and the frequency of mutations in the CNGA3 gene (encoding α subunit of the cone-specific cGMP-gated cation channel) was 23–33% in European populations. The aim of this study was to test the hypothesis that CNGA3 mutations are also responsible for congenital achromatopsia in Japanese patients. DNA from venous blood samples from a total of 14 patients from 13 Japanese pedigrees was prepared. Mutation screening of the CNGA3 gene was performed using direct sequencing and PCR-single-strand conformation polymorphism analysis. Compound heterozygous missense mutations (p.R436W and p.L633P, the latter of which was novel) were identified in one patient only, a 22-year-old female. Neither of these two mutations was found in 150 Japanese control individuals. The patient's parents and sister carried one of these mutations each but were not affected. No mutations in the CNGB3 or GNAT2 genes were identified in the patient. Clinically, best-corrected visual acuity was 0.1 in both eyes. No specific findings were obtained in funduscopy. Optical coherence topography revealed a normal foveal thickness but a 20% decrease in parafoveal thickness. Ganzfeld full-field electroretinograms (ERGs) showed normal responses in rod and mixed rod-plus-cone ERGs but no response in cone or 30-Hz flicker ERGs. Spectral sensitivity on a white background revealed a curve with only one peak at around 500 nm, which fits the absorption spectrum of human rhodopsin. L633, conserved among vertebrate orthologs of human CNGA3, is a hydrophobic residue forming part of the carboxy-terminal leucine zipper (CLZ) domain, which is functionally important in the mediation of intracellular interactions. To our knowledge, this is the first report of a Japanese complete achromat with CNGA3 mutations, and of any patient with a missense mutation within the CLZ domain. The outcome suggests low frequency (7%, 1/14) of CNGA3 mutations in Japanese patients.

scholarly journals Genome-wide scan identifies novel modifier loci of acromegalic phenotypes for isolated familial somatotropinoma

2009 ◽  
Vol 16 (3) ◽  
pp. 1057-1063 ◽  
Author(s):  
S K Khoo ◽  
R Pendek ◽  
R Nickolov ◽  
D C Luccio-Camelo ◽  
T L Newton ◽  
...  
Keyword(s):  
Pituitary Adenoma ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Incomplete Penetrance ◽  
Interacting Protein ◽  
Mutation Status ◽  
Aryl Hydrocarbon ◽  
Genome Wide ◽  
A Genome ◽  
Genome Wide Scan

Isolated familial somatotropinoma (IFS) accounts for 18% of familial isolated pituitary adenoma (FIPA) cases. Recently, germline mutations of the aryl hydrocarbon receptor-interacting protein gene (AIP) have been found in families with pituitary adenoma predisposition, FIPA, and IFS. In this study, we investigate the AIP mutation status and perform a genome-wide scan to search for the modifier regions of acromegalic phenotypes in an IFS family of 31 aborigines from Borneo. Complete endocrine diagnosis and data could not be collected due to logistical and cultural reasons. AIP mutation screening was carried out by direct sequencing and the genome-wide scan was performed using 400 microsatellites. Non-parametric linkage analysis was performed to obtain the logarithm of odds (LOD) scores. A novel AIP frameshift mutation in exon 4 (c.500delC) (p.P167HfsX3) was identified in all members with acromegalic features, as well as in 15 members without acromegalic features, revealing incomplete penetrance of AIP. The data showed that patients with the same mutation may express acromegalic features of differing severity, suggesting the existence of modifier genes. The highest LOD score of 2.2 was obtained near D19S571 (19q13.41). We also found weak linkages on chromosomes 3q28, 8q12.1, and 21q22.13, with LOD scores of 1.1, 1.8, and 1.4 respectively. Our results show the first genome-wide scan that identifies novel modifier loci for acromegalic phenotypes in an IFS family. Identification of modifier loci may provide further insight into the disease mechanism and explain the clinical variability observed in its patients.

scholarly journals ROLE OF THE NOTCH1 GENE IN FORMATION OF AORTIC ANEURYSM

2018 ◽  
pp. 53-59
Author(s):  
O. V. Irtyuga ◽  
O. A. Freilichman ◽  
D. S. Krivonosov ◽  
A. B. Malashicheva ◽  
S. I. Tarnovskaya ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Protein Protein Interactions ◽  
Highly Pathogenic ◽  
Targeted Mutation ◽  
Aortic Pathology ◽  
Inborn Defects ◽  
The Impact ◽  
Inborn Defect

Aim. To evaluate the impact of genetics in development of thoracic aneurysm in patients with tricuspid valve (TAV) and bicuspid aortic valve (BAV) based on the analysis and search for mutations in NOTCH1.Material and methods. In the study, 60 patients included with the dilation of thoracic aorta more than 40 mm and 200 patients with no aortic pathology, included in the comparison group. All patients underwent echocardiographic assessment on Vivid 7 (GE, USA) equipment, by standard protocol. For molecular genetics we utilized the strategy of targeted mutation screening, including the analysis of 10 of 34 exones of the gene NOTCH1, performed with the direct sequencing.Results. Patients with BAV were younger than those with no inborn defect. Arterial hypertension was verified only in every second BAV patient. Also, maximal rates of blood pressure were significantly lower in patients with inborn defects (р<0,02). As a result of genetic analysis in the studied group, in 9 patients with inborn defect and 2 patients with TAV there were 10 variants found of aminoacid replacement in 6 among 10 analyzed exones, of those 5 replacements — synonimic, and the mutation S2449R was found first time. Mutations P1227S, E1305K, R1279H and D1267N were found at the site of Notch1 protein binding with DLL4, of those 3 are highly pathogenic, that could influence the protein-protein interactions Notch1 with DLL4 leading to formation on aneurysm.Conclusion. Mutations P1227S D1267, E1305K in the gene NOTCH1, being highly pathogenic, may lead to the changes of protein functioning via Notch signalling disorder, that is more characteristic for BAV patients.

Mutations of the TP53 Gene Occur in 13.4% of Acute Myeloid Leukemia and Are Strongly Associated with a Complex Aberrant Karyotype.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1913-1913
Author(s):  
Claudia Schoch ◽  
Frank Dicker ◽  
Hannes Herholz ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Tp53 Mutation ◽  
Fusion Gene ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Molecular Genetic ◽  
Treatment Strategies ◽  
Normal Karyotype ◽  
Tp53 Gene ◽  
Trisomy 8 ◽  
Tp53 Mutations

Abstract The TP53 gene is the most frequently mutated gene in human tumors identified so far. In a prior study we demonstrated that 78% of AML with complex aberrant karyotype show a mutation of the TP53 gene. The aim of this study was to determine the frequency of TP53 mutations in an unselected cohort of AML and to analyze the relation to cytogenetic and molecular genetic aberrations. In total 149 AML cases were examined by chromosome banding analysis and screened for FLT3-length mutations (FLT3-LM), MLL partial tandem duplication (MLL-PTD), NPM1 mutations, and TP53 mutations. The cohort included cases with t(8;21) (n=10), t(15;17) (n=6), inv(16) (n=4), 11q23/MLL-rearrangement (n=6), trisomy 8 sole (n=13), AML with normal karyotype (n=46), AML with complex aberrant karyotype defined as showing 3 and more clonal abnormalities but no balanced rearrangement leading to a leukemia specific fusion gene (n=26), and AML with other abnormalities (n=38). FLT3-LM were observed in 21, MLL-PTD in 4, and NPM1-Mutations in 26 cases. TP53 mutation screening of exons 3–9 was performed by denaturing high performance liquid chromatography (DHPLC). All mutations detected were verified by direct sequencing. Overall, TP53 mutations were detected in 20 of the 149 cases (13.4%). Within this cohort of TP53 mutated cases, coincidences of FLT3-LM and MLL-PTD, respectively, with TP53 mutation were detected in one case each. A complex aberrant karyotype was present in 17 of 20 cases (85%) with TP53 mutation. The remaining 3 cases with TP53 mutation showed a normal karyotype, a trisomy 8, and t(8;21) as the sole abnormality, respectively. Therefore, we confirmed a high incidence of TP53 mutations in AML with complex aberrant karyotype (17/26, 65.4%). On the other hand TP53 mutations are very rare in AML without a complex aberrant karyotype (3/123, 2.4%). Furthermore, we divided AML with complex aberrant karyotype into two subgroups:AML with “typical” complex aberrant karyotype showing a deletion of at least one of the following regions: 5q31, 7q31, 17p13 (definition according to Schoch et al. GCC, 2005) andAML with “untypical” complex aberrant karyotype comprizing all others. Interestingly, the frequency of TP53 mutations within the “typical” complex aberrant karyotype group was 75% (15/20) while in the “untypical” group it was 33% (2/6) (p=0.138). In conclusion, the overall incidence of TP53 mutations is low in AML. TP53 mutations are highly associated with AML and complex aberrant karyotype and occur very infrequently in all other cytogenetic subgroups (p<0.001). They occur frequently in particular in the subgroup showing a typical pattern of chromosomal deletions (5q, 7q, 17p). TP53 mutations might explain in part the chemoresistance of AML with complex aberrant karyotype. In addition to cytogenetics a rapid diagnostic screening for TP53 mutations could be a valuable tool to identify a subgroup of AML with poor prognosis. This would allow the early assignment of patients to alternative treatment strategies using also options targeting the TP53 pathway.

Detection of BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia Patients Treated with Tirosin-Kinase Inhibitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4261-4261
Author(s):  
Gonzalo Manrique ◽  
Roberta Bittencout ◽  
Verónica Pérez ◽  
Vanesa Sholl ◽  
Monica Cappetta ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Low Cost ◽  
Mutation Screening ◽  
Point Mutations ◽  
Kinase Domain ◽  
Rapid Screening ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Allele Specific

Abstract Background. Point mutations in the kinase domain (KD) of the BCR-ABL are the most frequent mechanism of drug resistance in CML patients treated with kinase inhibitors (TKI). More than 80 mutations with different frequency and clinical significance have been reported. One of them, the T315I confers resistance to all TKIs available. The detection of mutations in KD allows early identification of high-risk patients and therefore guides clinical therapy decisions. Aim. To assess the mutation status of a group of CML pts resistant to TKI from Uruguay (n=35) and Brazil (n=30). Methods. KD mutation screening was performed by RT-PCR and direct sequencing according to Branford et al. (2002). Additionally, we developed a rapid, specific, sensitive and low cost allele specific (AS)-RT-PCR assay to identify T315I, using Branford’s KD amplification primers in combination with an allele specific primer for the T315I point mutation detection. BCR-ABL transcript levels were also measured by RQ-PCR according to international recommendations. Results and Discussion. RT-PCR and direct sequencing analyses performed in all pts showed the presence of T315l mutation in 3/65 cases. Other 11 showed the alternative mutations Y253H (n=2), E450A, G250E (n=2), E459K (n=2), E450G, F317L (n=2) and E255K; and the remaining 55 showed no mutations in the ABL KD. All 65 samples together with cDNA from 15 non-resistant CML pts and 10 cDNA from non-CML were analyzed by AS-RT-PCR assay for T315l mutation in order to validate the method. T315l was identified in the 3 samples in which the mutation was previously detected by direct sequencing and in 1 pt that had been classified as KD mutation negative. This result was then confirmed by direct sequencing of the AS-PCR product. T315 was neither detected in samples positive for other mutations nor in samples of non-resistant CML and non-CML patients, supporting the specificity of the method. Assessment of the sensitivity of the AS-RT-PCR was performed on serial dilutions experiments using RNA from T315 positive pt into RNA from CML-T315l negative pt, showing that the T315I mutation was detectable to a level of 0.01 % by AS-PCR, while through direct sequencing method the sensitivity was 10–20%. The prevalence of mutations in our study was 15/65 (23%). Conclusions. Our results showed that the AS-RT-PCR described here is a convenient and easy tool to be used in a clinical routine laboratory for rapid screening for BCR-ABL T315. This, together with direct sequencing, constitutes a suitable approach for CML resistance monitoring and therapeutic choice.

Linkage mapping of a nonspecific form of X-linked mental retardation (MRX53) in a large Pakistani family

2001 ◽  
Vol 100 (1) ◽  
pp. 62-65 ◽  
Author(s):  
Wasim Ahmad ◽  
Sara Noci ◽  
Mohammad Faiyaz ul Haque ◽  
Tiziana Sarno ◽  
Paolo Aridon ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Linkage Mapping ◽  
Pakistani Family

scholarly journals Identification of Novel ARSA Mutations in Chinese Patients with Metachromatic Leukodystrophy

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Li Chen ◽  
Huifang Yan ◽  
Binbin Cao ◽  
Ye Wu ◽  
Qiang Gu ◽  
...  
Keyword(s):  
Metachromatic Leukodystrophy ◽  
Direct Sequencing ◽  
Chinese Patients ◽  
Insertion Mutation ◽  
Inherited Disease ◽  
Wild Type ◽  
Novel Mutations ◽  
Genetic Characteristics ◽  
Developmental Problems ◽  
Polymerase Chain

Objective. Metachromatic leukodystrophy (MLD) is an inherited disease caused by a deficiency of the enzyme arylsulfatase A (ARSA) that leads to severe physiologic and developmental problems. Our study is aimed at elucidating the clinical and genetic characteristics of Chinese MLD patients. Methods. Clinical data of 21 MLD patients was collected. All coding exons of ARSA and their flanking intronic sequences were amplified by polymerase chain reaction and subjected to direct sequencing. Results. All 21 patients were diagnosed with MLD clinically and genetically, out of which 17 patients were late infantile and 4 were juvenile types. A total of 34 ARSA mutations, including 28 novel mutations (22 missense, 1 splicing, 1 nonsense, 3 small insertions, and 1 small deletion mutation) and 6 known mutations (5 missense and 1 small insertion mutation), were identified. Prenatal diagnosis was performed for four pedigrees. One fetus was a patient, two fetuses were carriers, and two were wild type. Conclusions. The present study discovered 28 novel ARSA mutations and widely expanded the mutation spectrum of ARSA. Four successful prenatal diagnoses provided critical information for MLD families.

scholarly journals Calreticulin and JAK2 Exon 12 Mutation Screening in Patients with Myeloproliferative Neoplasms’ in Jeddah Region, Saudi Arabia

2021 ◽  
pp. 420-428
Author(s):  
Heba Alkhatabi ◽  
Heyam Abdulqayoom ◽  
Raed Alserihi ◽  
Raed Felimban ◽  
Aisha Elaimi ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Pcr Analysis ◽  
Allele Specific ◽  
Pcr Method ◽  
Calr Mutations ◽  
The Impact

Background: The JAK2 V617F mutation’s discovery has largely facilitated the comprehension of the myeloproliferative neoplasms’(MPNs) pathogenesis. In recent times, calreticulin (CALR) mutations have been detected in patients with JAK2V617F negative primary myelofibrosis (PMF), and essential thrombocythemia (ET). Methods: This study analyzed the impact of JAK 2 Exon 12 and CALR common mutations in 65 patients with JAK2V617F negative MPN from the Jeddah region. An allele-specific polymerase chain reaction (PCR) method was used to screen four common mutations on Exon 12 and direct sequencing and PCR analysis were utilized to screen all patients for CALR. Results: None of the patients were positive for the Exon 12 mutation and eight patients were positive for CALR mutations. Conclusions: This is the first Saudi Arabian research that focused on screening CALR hotspot mutations and this mutation exists. This fact highlights the importance of implementing diagnostic screening of CALR on MPN patients, in general, and patients with high platelet count, in particular. Further screening of other predisposing genetic markers might facilitate the identification of an important genetic variant, which could aid in the understanding of disease pathogenesis.

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