The spectrum of genetic changes in the RB1 gene in a group of Russian retinoblastoma patients

2021 ◽  
pp. 11-20
Author(s):  
Е.А. Алексеева ◽  
В.М. Козлова ◽  
О.В. Бабенко ◽  
Т.Л. Ушакова ◽  
Т.П. Казубская ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Genetic ◽  
Genetic Diagnosis ◽  
Point Mutations ◽  
Missense Mutations ◽  
Rb1 Gene ◽  
Genetic Changes ◽  
Parallel Sequencing ◽  
Large Deletions ◽  
Efficient Detection

Введение. Ретинобластома - злокачественная опухоль детского возраста, причиной которой является биаллельная инактивация гена RB1. Ранняя молекулярно-генетическая диагностика ретинобластомы необходима как для адекватного выбора алгоритма лечения пациента с такой опухолью, так и для медико-генетического консультирования семьи. Цель: охарактеризовать частоту и спектр мутаций в гене RB1 у российских больных с ретинобластомой. Методы. Исследование проведено на материале ДНК лимфоцитов крови, полученном от 492 больных с ретинобластомой. Скрининг точковых мутаций, малых инсерций/делеций в гене RВ1 осуществляли методом полупроводникового высокопроизводительного параллельного секвенирования. Исключение протяженных делеций в гене RВ1 проводили методом MLPA. Результаты. Исследовано 492 неродственных пациента с ретинобластомой, среди которых 38,2% (188/492) с билатеральной формой заболевания и 61,8% (304/492) - с унилатеральной. В группе больных с билатеральной формой ретинобластомы герминальная мутация обнаружена у 96,8% (182/188) пациентов, в группе больных с унилатеральной формой - у 16,4% (50/304). Суммарно в гене RB1 в исследованной группе пациентов обнаружено 339 мутаций: 232 - герминальных и 107 - соматических. Выявлен практически полный спектр молекулярных изменений, включающий нонсенс-мутации - 37,5% (127/339), миссенс-мутации - 5,3% (18/339), мутации, приводящие к сдвигу рамки считывания - 18,9% (64/339), мутации сайтов сплайсинга - 13,9% (47/339) и протяженные делеции - 24,5% (83/339). Выводы. Применение глубокого высокопроизводительного параллельного секвенирования и метода MLPA позволяет эффективно выявлять молекулярно-генетические изменения в гене RB1. Типы мутаций, обнаруженные в исследованной группе, их частота и распределение совпадают с результатами исследователей из других стран. Background. Retinoblastoma is a childhood malignant tumor caused by biallelic inactivation of the RB1 gene. Early molecular genetic diagnosis of retinoblastoma is necessary both for an adequate choice of an algorithm for treating a patient, and for competent medical genetic counseling of the family Objective. To establish the frequency and spectrum of mutations in the RB1 gene in the group of patients with retinoblastoma. Methods. The study was carried out on the DNA of blood lymphocytes from 492 patients with retinoblastoma. Screening of point mutations, small insertions/deletions in the RB1 gene was performed by semiconductor high-throughput parallel sequencing. Exclusion of gross deletions in the RB1 gene was performed by MLPA. Results. 492 unrelated patients with retinoblastoma were studied, including 38.2% (188/492) with bilateral form and 61.8% (304/492) with unilateral form. In the group of patients with bilateral retinoblastoma, germline mutation was found in 96.8% (182/188) patients, and in the group of unilateral patients, in 16.4% (50/304). In total, the RB1 gene in the studied group of patients 339 mutations were found, 232 germline and 107 somatic. An almost complete spectrum of molecular changes was revealed, including nonsense mutations, 37.5% (127/339); missense mutations, 5.3% (18/339); frame shift mutations, 18.9% (64 / 339); splice site mutations, 13.9% (47/339); and large deletions, 24.5% (83/339). Conclusion. The use of deep high-throughput parallel sequencing and the MLPA method allows efficient detection of molecular genetic changes in the RB1 gene. The types of mutations found in the studied group, their frequency and distribution are the same as the results of researchers in other countries.

Somatic mosaicism in sporadic retinoblastoma

2021 ◽  
pp. 9-18
Author(s):  
Е.А. Алексеева ◽  
К.О. Карандашева ◽  
О.В. Бабенко ◽  
В.М. Козлова ◽  
Т.Л. Ушакова ◽  
...  
Keyword(s):  
Genetic Counseling ◽  
High Throughput ◽  
Mutant Allele ◽  
De Novo ◽  
The Body ◽  
Null Alleles ◽  
Medical Genetic ◽  
Rb1 Gene ◽  
Parallel Sequencing ◽  
Depth Analysis

Введение. Спорадическая ретинобластома развивается в результате мутаций de novo в обоих аллелях гена RB1 в клетках сетчатки глаза. При спорадической ретинобластоме первоначальная мутация в гене RB1 нередко является мозаичной, то есть образуется в постзиготической ранней эмбриональной клетке, что приводит к неравномерному распределению мутантных клонов между различными тканями организма. Возможность идентифицировать мозаичный вариант мутации в гене RB1 имеет значение как для медико-генетического консультирования, так и для клинического ведения пациентов, поскольку мозаицизм влияет на развитие клинической картины заболевания, риск развития опухоли в другом глазу и других опухолей и на риск передачи мутации следующему поколению. Цель: установить частоту и спектр постзиготических мозаичных мутаций в гене RB1 в выборке больных со спорадической ретинобластомой, определить содержание мутантного аллеля в образцах с мозаицизмом. Метод. Исследование проведено на материале ДНК лимфоцитов крови больных со спорадической ретинобластомой. Скрининг точковых мутаций, малых инсерций/делеций в гене RВ1 осуществляли методом полупроводникового высокопроизводительного параллельного секвенирования (ВПС). Исключение протяженных делеций в гене RВ1 проводили методом MLPA. Для поиска мозаичных мутаций с очень низким содержанием (менее 10%) мутантного аллеля был разработан и проведен углубленный анализ данных ВПС, основанный на биоинформатических и статистических подходах. Для верификации выявленных мозаичных патогенных мутаций использовали секвенирование ДНК по Сэнгеру. Результаты. В исследованной выборке больных со спорадической унилатеральной формой ретинобластомы мозаичные мутации встречаются чаще, чем при спорадической билатеральной форме; различия статистически достоверны. В то же время, частоты мозаичных мутаций с высокой и низкой представленностью мутантных аллелей между группами больных с унилатеральной и билатеральной ретинобластомой достоверно не различаются. Все мозаичные мутации, представлены нуль-аллелями; мозаичных миссенс-мутаций в нашей выборке не обнаружено. Не выявлено мозаичных мутаций в 1-м и 2-м экзонах гена RB1, расположенных проксимальнее альтернативного промотора, импринтинг которого определяет пенетрантность мутаций в зависимости от родительского происхождения мутантного аллеля. Заключение. Применение глубокого ВПС в сочетании с усовершенствованным алгоритмом анализа результатов, направленным на выявление мозаичных мутаций, повышает эффективность ДНК-диагностики ретинобластомы, способствуя совершенствованию медико-генетического консультирования и лечения больных. Background. Sporadic retinoblastoma develops as a result of de novo mutations in both alleles of the RB1 gene. Often in sporadic retinoblastoma, the initial mutation in RB1 is mosaic, that is, it is formed in a postzygotic, early embryonic cell, which leads to an uneven distribution of mutant clones between different tissues of the body. The ability to identify a mosaic variant of a mutation in the RB1 gene is important for both medical genetic counseling and clinical management of patients, since mosaicism affects the development of the clinical picture of the disease, the risk of developing a tumor in the other eye, as well as other tumors, and the risk of mutation transmission to the next generation. Aim: to establish the frequency and spectrum of somatic mosaic mutations in the RB1 gene in patients with sporadic retinoblastoma and to quantify the content of the mutant allele in cases with mosaicism. Methods. The study was carried out on the DNA of blood lymphocytes from patients with sporadic retinoblastoma. Screening of point mutations, small insertions/deletions in the RB1 gene was performed by semiconductor high-throughput parallel sequencing (NGS). Exclusion of gross deletions in the RB1 gene was performed by MLPA. To search for mosaic mutations with a very low representation (less than 10%) of the mutant allele, an in-depth analysis of the NGS data was developed an in-house algorithm based on bioinformatic and statistical approaches. To verify mosaic pathogenic mutations identified with NGS, Sanger sequencing was used. Results. Mosaic mutations were found more common among patients with sporadic unilateral form of retinoblastoma than in those with sporadic bilateral form; the differences are statistically significant. At the same time, the frequencies of mosaic mutations with a high and low representation of mutant alleles between the groups of patients with unilateral and bilateral retinoblastoma did not differ significantly. All mosaic mutations are null alleles; mosaic missense mutations were not found in our patients’ cohort. No mosaic mutations were detected in the 1st and 2nd exons of the RB1 gene, located proximal to the alternative promoter, the imprinting of which determines the penetrance of mutations depending on the parental origin of the mutant allele. Conclusion. The use of deep high-throughput parallel sequencing in combination with an improved algorithm for analyzing the NGS results, aimed at identifying mosaic mutations, increases the efficiency of DNA diagnostics of retinoblastoma, contributing to the improvement of medical genetic counseling and treatment of patients.

scholarly journals Assessment of the F9 genotype-specific FIX inhibitor risks and characterisation of 10 novel severe F9 defects in the first molecular series of Argentinian patients with haemophilia B

2013 ◽  
Vol 109 (01) ◽  
pp. 24-33 ◽  
Author(s):  
Liliana Carmen Rossetti ◽  
Miguel Martín Abelleyro ◽  
Miguel Candela ◽  
Raúl Pérez Bianco ◽  
Miguel de Tezanos Pinto ◽  
...  
Keyword(s):  
Point Mutations ◽  
Bioinformatic Analysis ◽  
Factor Ix ◽  
Causative Mutation ◽  
Control Analysis ◽  
Missense Mutations ◽  
Novel Mutations ◽  
Haemophilia B ◽  
Large Deletions ◽  
Genotype Frequencies

SummaryIn haemophilia B (HB) (factor IX [FIX] deficiency), F9 genotype largely determines clinical phenotype. Aimed to characterise Argentinian families with HB, this study presents F9 genotype frequencies and their specific FIX inhibitor risk and 10 novel F9 mutations. Ninety-one DNA samples from HB patients and relatives were subjected to a new scheme: a primary screen for large deletions, a secondary screen for point mutations using conformation sensitive gel electrophoresis, DNA-sequencing and bioinformatic analysis. Our unbiased HB population (N=52) (77% with severe, 11.5% moderate and 11.5% mild HB) showed 32 missense (61.5%), including three novel mutations predicting specific structural/functional defects in silico, seven nonsense (13.5%) (one novel), five large deletions, four splice including three novel mutations affecting predicted splicing scores, three indels (two novel) and one Leiden mutation. Our comprehensive HB population included five patients with long-lasting FIX inhibitors: three nonsense (p.E35* (novel), p.R75*, p.W240*) and two entire-F9 deletions. Another patient with an indel (p.A26Rfs*14) developed transient inhibitors. A case-control analysis, based on our global prevalence of 3.05% for developing inhibitors in HB revealed that missense mutations were associated with a low risk odds ratio (OR) of 0.05 and a prevalence of 0.39%, whereas nonsense and entire-F9 deletions had significantly higher risks (OR 11.0 and 32.7) and prevalence (14.3% and 44.5%, respectively). Our cost-effective practical approach enabled identification of the causative mutation in all 55 Argentine families with HB, analysis of the molecular pathology of novel F9 defects and determination of mutation-associated FIX inhibitor risks.

scholarly journals Genetic Diversity of the Q Fever Agent, Coxiella burnetii, Assessed by Microarray-Based Whole-Genome Comparisons

2006 ◽  
Vol 188 (7) ◽  
pp. 2309-2324 ◽  
Author(s):  
Paul A. Beare ◽  
James E. Samuel ◽  
Dale Howe ◽  
Kimmo Virtaneva ◽  
Stephen F. Porcella ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Point Mutations ◽  
Open Reading Frames ◽  
Cellular Functions ◽  
Genetic Changes ◽  
Large Deletions ◽  
Reference Isolate ◽  
Virulence Potential

ABSTRACT Coxiella burnetii, a gram-negative obligate intracellular bacterium, causes human Q fever and is considered a potential agent of bioterrorism. Distinct genomic groups of C. burnetii are revealed by restriction fragment-length polymorphisms (RFLP). Here we comprehensively define the genetic diversity of C. burnetii by hybridizing the genomes of 20 RFLP-grouped and four ungrouped isolates from disparate sources to a high-density custom Affymetrix GeneChip containing all open reading frames (ORFs) of the Nine Mile phase I (NMI) reference isolate. We confirmed the relatedness of RFLP-grouped isolates and showed that two ungrouped isolates represent distinct genomic groups. Isolates contained up to 20 genomic polymorphisms consisting of 1 to 18 ORFs each. These were mostly complete ORF deletions, although partial deletions, point mutations, and insertions were also identified. A total of 139 chromosomal and plasmid ORFs were polymorphic among all C. burnetii isolates, representing ca. 7% of the NMI coding capacity. Approximately 67% of all deleted ORFs were hypothetical, while 9% were annotated in NMI as nonfunctional (e.g., frameshifted). The remaining deleted ORFs were associated with diverse cellular functions. The only deletions associated with isogenic NMI variants of attenuated virulence were previously described large deletions containing genes involved in lipopolysaccharide (LPS) biosynthesis, suggesting that these polymorphisms alone are responsible for the lower virulence of these variants. Interestingly, a variant of the Australia QD isolate producing truncated LPS had no detectable deletions, indicating LPS truncation can occur via small genetic changes. Our results provide new insight into the genetic diversity and virulence potential of Coxiella species.

scholarly journals Advantages of high throughput parallel sequencing in detecting somatic mosaicism in sporadic retinoblastoma

2020 ◽  
pp. 6-7
Author(s):  
Е.А. Алексеева ◽  
О.В. Бабенко ◽  
В.М. Козлова ◽  
Т.Л. Ушакова ◽  
Т.П. Казубская ◽  
...  
Keyword(s):  
Family History ◽  
High Throughput ◽  
De Novo ◽  
Somatic Mosaicism ◽  
Allelic Frequency ◽  
De Novo Mutations ◽  
Rb1 Gene ◽  
Parallel Sequencing ◽  
Pathogenic Variants

Почти 80% случаев наследственной ретинобластомы не имеют семейного анамнеза и возникают в результате мутаций de novo в гене RB1. Методом высокопроизводительного параллельного секвенирования (ВПС) проведено молекулярно-генетическое обследование 208 неродственных больных со спорадической РБ, среди которых 145 пациентов с унилатеральной формой заболевания и 63 - с билатеральной. В группе пациентов с билатеральной РБ молекулярные изменения в гене RB1 обнаружены в 90,5% (57/63) случаев. У 4,8% (3/63) пациентов определен мозаичный вариант мутации в гене RB1. В группе пациентов с унилатеральной РБ молекулярные изменения в гене RB1 выявлены в 17,9% (26/145) случаев. Среди исследованных пациентов соматический мозаицизм выявлен в 9,0% (13/165) случаев. Применение ВПС позволяет точно определять аллельную частоту вариантов, что делает поиск соматического мозаицизма эффективным. Almost 80% of cases of hereditary retinoblastoma do not have a family history and arise as a result of de novo mutations in the RB1 gene. An NGS test was performed on 208 unrelated patients with sporadic RB, including 145 patients with a unilateral form and 63 patients with a bilateral one. In the group of patients with bilateral RB, pathogenic variants in the RB1 gene were detected in 90.5% (57/63) cases. In 4.8% (3/63) of patients, a mosaic variants were determined. In the group of patients with unilateral RB, changes in the RB1 gene were detected in 17.9% (26/145) cases. Among the examined patients, somatic mosaicism was detected in 9.0% (13/165) cases. NGS allows us to determine the allelic frequency of variants, which makes the search for somatic mosaicism effective.

Characterisation of 96 mutations in 128 unrelated severe haemophilia A patients from France

2006 ◽  
Vol 95 (04) ◽  
pp. 593-599 ◽  
Author(s):  
Christine Vinciguerra ◽  
Christophe Zawadzki ◽  
Yesim Dargaud ◽  
Gilles Pernod ◽  
Claire Berger ◽  
...  
Keyword(s):  
Factor Viii ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Severe Haemophilia ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Large Deletions ◽  
Fviii Inhibitor

SummaryDirect sequencing of the coding region of factor VIII (F8) gene was used to determine the mutations responsible for severe haemophilia A (FVIII<1%) in 128 unrelated haemophiliacs A, negative for intron 22 and intron 1 inversions. A mutation was found in 122/128 patients (95%). Ninety-six distinct mutations were identified in this cohort, 62 of these are novel. They consisted of deletions (7 large and 24 small deletions), insertions (n=9), associations of insertion/deletion (n=2), association of deletion/substitution (n=1), and single nucleotide substitutions (53 point mutations consisting of 31 missense, 20 nonsense, and 2 splicing mutations). Twenty-two patients had developed inhibitors, and among this subgroup 3 large deletions, 6 frameshift, 9 nonsense and 4 missense mutations were detected. For6 patients, among which one developed an anti-FVIII inhibitor, no mutations were detected in the coding and splicing regions of factor VIII gene. Different approaches of molecular modelling were performed in addition to familial linkage analysis to determine the pathophysiological responsibility of these novel missense mutations.

Clinical and molecular characterisation of 21 patients affected by quantitative fibrinogen deficiency

2015 ◽  
Vol 113 (03) ◽  
pp. 567-576 ◽  
Author(s):  
Rosanna Asselta ◽  
Manuela Platè ◽  
Michela Robusto ◽  
Munira Borhany ◽  
Ilaria Guella ◽  
...  
Keyword(s):  
Point Mutations ◽  
Compound Heterozygous ◽  
Molecular Characterisation ◽  
Missense Mutations ◽  
Large Deletions ◽  
Mutational Screening ◽  
Premature Termination Codons ◽  
Polypeptide Chains ◽  
Compound Heterozygous State

SummaryFibrinogen is a plasma glycoprotein mainly synthesised by hepatocytes and circulating as a 340-kDa hexamer consisting of two sets of three different polypeptide chains (Aα, Bβ, and γ, encoded by the FGA, FGB, and FGG gene, respectively). Congenital afibrinogenaemia and hypofibrinogenaemia are rare bleeding disorders characterised by abnormally low levels of functional and immunoreactive fibrinogen in plasma, associated with haemorrhagic manifestations of variable severity. While afibrinogenaemia is caused by mutations in the homozygous or compound heterozygous state in one of the three fibrinogen genes, hypofibrinogenaemia is generally due to heterozygous mutations, and is usually characterised by a milder phenotype. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations often affecting fibrinogen assembly and/or secretion. Here we report the clinical and molecular characterisation of 13 unrelated afibrinogenaemic and eight hypofibrino - genaemic patients, leading to the identification of 17 different mutations (10 hitherto unknown). All the newly-identified missense and splicing mutations were in vitro expressed to verify their pathogenic role. Our data increase the number of mutations causing quantitative fibrinogen deficiencies by about 7 %. The high number of private mutations identified in the analysed probands indicates that the full mutational screening of the three fibrinogen genes is still required for molecular diagnosis.

Whole-Transcriptome Sequencing In Chronic Myeloid Leukemia Reveals Novel Gene Mutations That May Be Associated with Disease Pathogenesis and Progression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 885-885 ◽  
Author(s):  
Simona Soverini ◽  
Angela Poerio ◽  
Alberto Ferrarini ◽  
Ilaria Iacobucci ◽  
Marco Sazzini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myeloid Leukemia ◽  
Massively Parallel Sequencing ◽  
Blast Crisis ◽  
Point Mutations ◽  
Digital Gene Expression ◽  
Massively Parallel ◽  
Missense Mutations ◽  
Parallel Sequencing ◽  
Whole Transcriptome

Abstract Abstract 885 Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) has always been regarded as a genetically homogeneous disease. However, the fact that a proportion of patients (pts), especially in the high Sokal risk setting, fail tyrosine kinase inhibitor therapy and progress to blast crisis (BC) suggests that a certain degree of heterogeneity exists. It can be hypothesized that genetic factors additional to the Ph+ chromosome may be present in these pts. To address this issue, we are currently using massively parallel sequencing to perform a qualitative and quantitative survey of the whole transcriptome of Ph+ CML cells at diagnosis and at progression to BC. Results are being integrated with genome-wide search for copy number alterations by Affymetrix SNP 6.0 arrays. We used a Solexa Illumina Genome Analyzer to scan the transcriptome of a CML patient at the time of diagnosis, at the time of remission (major molecular response) and at the time of progression from chronic phase (CP) to lymphoid blast crisis (BC). Both custom scripts and published algorithms were used for read alignment against the human reference genome, for single nucleotide variant (SNV) calling, for identification of alternative splicings and fusion transcripts, and for digital gene expression profiling. Comparison of the SNVs identified in the diagnosis and relapse samples with the SNVs detected in the remission sample – representing inherited sequence variants not specific for the Ph+ clone – allowed the identification of eight missense mutations at diagnosis affecting the coding sequences of AMPD3 (encoding adenosine monophosphate deaminase 3), SUCNR1 (succinate receptor 1), FANCD2 (Fanconi anemia, complementation group D2), INCENP (inner centromere protein), BSPRY (B-box and SPRY domain containing), HEXDC (hexosaminidase containing), NUDT9 (ADP-ribose diphosphatase) and KIAA2018 (encoding a protein with predicted DNA binding and transcriptional regulation activity) genes. Six of these mutations (FANCD2, INCENP, BSPRY, HEXDC, NUDT9) were also detected in the Ph+ clone re-emerged at the time of disease progression, together with seven additional missense mutations affecting the coding sequences of IDH2 (isocitrate dehydrogenase isoform 2), DECR1 (2,4-dienoyl CoA reductase 1), C4Orf14 (mitochondrial nitric oxide synthase), MRM1 (mitochondrial rRNA methyltransferase 1), PRKD2 (protein kinase D2), TCHP (mitostatin) and ABL1 genes. Digital gene expression analysis showed downregulation of SUCNR1, that might be a consequence of the P292A mutation we detected. IDH2, MRM1, AMPD3, and KIAA2018 mutations were found in additional pts. The IDH2 R140Q mutation was detected in 3/75 (4%) myeloid BC, 1/31 (3.2%) lymphoid BC, 0/34 Ph+ ALL and 0/23 Philadelphia-negative (Ph-) ALL pts. The MRM1 C120S mutation was found in 6/70 (9%) additional BC pts (2 lymphoid and 4 myeloid). AMPD3 and KIAA2018 genes were found to harbour the same point mutations (N334S and S1818G, respectively) in 1 out of 20 additional CP patients analyzed. Massively parallel sequencing of the sample collected at diagnosis also revealed that the Bcr-Abl kinase domain was already harbouring point mutations at low levels (E308D, A344G, R386S) but not the T315I that was selected at the time of disease progression. Point mutations in untraslated regions where miRNAs are known to bind were also detected, and are currently under validation. Digital gene expression profiling comparing progression to diagnosis showed significant expression changes including upregulation of 134 genes and downregulation of 88 genes. In particular, we observed an upregulation of the B-cell developmental factor PAX5, its interactor Lef-1 and its targets IRF4, BLNK, Bik, EBF1, CD79A, CD79B, CD19, VpreB1, VpreB3, BOB1, RAG1 and RAG2; upregulation of PAX9; upregulation of WNT3A, WNT9A, GLI3 and downregulation of SFRP1, resulting in aberrant activation of the Wnt signalling pathway. In summary, our preliminary data highlighted putative key genes whose deregulation may be recurrent in a subset of CML patients and may be linked to disease pathogenesis or progression. Their actual role in CML is currently being exlored. Massively parallel sequencing of additional patients is ongoing. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants. Disclosures: Baccarani: NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

scholarly journals The effect of parental origin of RB1 mutations in hereditary retinoblastoma with low penetrance

2019 ◽  
pp. 21-28
Author(s):  
Е.А. Алексеева ◽  
О.В. Бабенко ◽  
В.М. Козлова ◽  
Т.Л. Ушакова ◽  
Т.П. Казубская ◽  
...  
Keyword(s):  
Germline Mutation ◽  
High Performance ◽  
Asymptomatic Carrier ◽  
Molecular Genetic ◽  
Point Mutations ◽  
Clinical Signs ◽  
Parental Origin ◽  
Incomplete Penetrance ◽  
Rb1 Gene ◽  
Variable Expressivity

Актуальность. При наследственной ретинобластоме герминальная мутация в одном из аллелей гена RB1 обусловливает предрасположенность к заболеванию и его семейную передачу. Наследственная ретинобластома манифестирует в более раннем возрасте по сравнению со спорадической формой и носит в большинстве случаев мультифокальный и билатеральный характер. Однако некоторые семьи с ретинобластомой (два и более носителя одинаковой герминальной мутации в родословной) демонстрируют более мягкий фенотип с неполной пенетрантностью (у части носителей герминальной мутации заболевание не развивается) и вариабельной экспрессивностью (одинаковая мутация у разных членов семьи может проявляться уни- или билатеральной формой заболевания). Выявление низкопенетрантных мутаций в гене RB1 и изучение характера их наследования способствует пониманию механизмов, лежащих в основе развития наследственной ретинобластомы с низкой пенетрантностью, и крайне важно как для дальнейшего расширения знаний о молекулярной генетике ретинобластомы, так и для медико-генетического консультирования и последующего клинического ведения семей с такой формой заболевания. Цель. Установить спектр генетических нарушений в гене RB1 у больных с наследственной ретинобластомой с неполной пенетрантностью и вариабельной экспрессивностью в выборке российских пациентов и определить влияние родительского происхождения мутации в RB1 на её фенотипическое проявление. Методы. Методом высокопроизводительного параллельного секвенирования проведено молекулярно-генетическое обследование 332 неродственных больных с ретинобластомой. Для верификации выявленных точковых мутаций и анализа их сегрегации в родословных использовали секвенирование по Сэнгеру. Результаты. В группе пациентов без семейного анамнеза ретинобластомы у 3,5% больных определен наследственный характер заболевания, при котором один из родителей являлся бессимптомным носителем герминальной мутации в гене RB1. Выявлено 10 низкопенетрантных мутаций в гене RB1 и установлен спектр мутаций, приводящих к развитию наследственной ретинобластомы с низкой пенетрантностью. В 91,7% случаев наследственной ретинобластомы с низкой пенетрантностью мутантный аллель получен пробандом от отца, у которого отсутствовали клинические признаки заболевания или наблюдалась более легкая форма. Выводы. Полученные результаты подтверждают ранее высказанные предположения, что низкопенетрантные герминальные мутации в гене RB1, унаследованные от отца, чаще приводят к развитию ретинобластомы, и в более тяжелой форме по сравнению с таковыми, унаследованными от матери. Background. In hereditary retinoblastoma, a germline mutation in one of the alleles of the RB1 gene causes a predisposition to the disease and its transmission within the pedigree. Hereditary retinoblastoma manifests at an earlier age compared with the sporadic form and in most cases is multifocal and bilateral. However, some families with retinoblastoma (two or more carriers of the same germline mutation in the pedigree) exhibit a milder phenotype with incomplete penetrance (some carriers of the germline mutation are asymptomatic) and variable expressivity (the same mutation in different family members may manifest as either a unilateral or bilateral form). Identification of low-penetrant mutations in the RB1 gene and studying their inheritance in pedigrees contributes to understanding the mechanisms underlying the development of retinoblastoma with low penetrance. It is extremely important both for further expansion of knowledge in the field of molecular genetics of retinoblastoma, and for competent genetic counseling and subsequent clinical management of families with this form of the disease. Objective. To establish the spectrum of genetic disorders in the RB1 gene in Russian patients with hereditary retinoblastoma with incomplete penetrance and variable expressivity and to determine the effect of the parental origin of the RB1 mutation on its phenotypic manifestation. Methods. Using high-performance parallel sequencing, a molecular genetic survey of 332 unrelated patients with retinoblastoma was performed. Sanger sequencing was used to verify the identified point mutations and analyze their segregation in pedigrees. Results. In the group of patients without a family history of retinoblastoma, in 3.5% the hereditary nature of the disease was determined, where one of the parents was an asymptomatic carrier of a germline mutation in the RB1 gene. Ten low-penetrant mutations in the RB1 gene were identified. In 91.7% of cases of hereditary retinoblastoma with low penetrance, the mutant allele was obtained by a proband from a father with no clinical signs of the disease or with a milder form. Conclusion. The results confirm the previously suggested assumptions that low-penetrant germline mutations in the RB1 gene inherited from the father more often lead to the development of retinoblastoma, and in a more severe form than those inherited from the mother.

scholarly journals Comprehensive One-Step Molecular Analyses of Mitochondrial Genome by Massively Parallel Sequencing

2012 ◽  
Vol 58 (9) ◽  
pp. 1322-1331 ◽  
Author(s):  
Wei Zhang ◽  
Hong Cui ◽  
Lee-Jun C Wong
Keyword(s):  
Mitochondrial Genome ◽  
Sensitivity And Specificity ◽  
Massively Parallel Sequencing ◽  
Point Mutations ◽  
Massively Parallel ◽  
Parallel Sequencing ◽  
Molecular Analyses ◽  
Mtdna Mutations ◽  
Large Deletions ◽  
Detection And Quantification

Abstract BACKGROUND Mitochondrial diseases are clinically and genetically heterogeneous, with variable penetrance, expressivity, and differing age of onset. Disease-causing point mutations and large deletions in the mitochondrial genome often exist in a heteroplasmic state. Current molecular analyses require multiple different and complementary methods for the detection and quantification of mitochondrial DNA (mtDNA) mutations. We developed a novel approach to analyze the mtDNA in 1 step. METHODS The entire human mitochondrial genome was enriched by a single amplicon long-range PCR followed by massively parallel sequencing to simultaneously detect mtDNA point mutations and large deletions with heteroplasmic levels of the mutations and variants quantified. QC samples were designed and analyzed along with each sample. A total of 45 samples were analyzed for the evaluation of analytic sensitivity and specificity. RESULTS Our analysis demonstrated 100% diagnostic sensitivity and specificity of base calls compared to the results from Sanger sequencing. The deep coverage allowed the detection and quantification of heteroplasmy at every single nucleotide position of the 16 569-bp mitochondrial genome. Moreover, the method also detected large deletions with the breakpoints mapped. CONCLUSIONS This “deep” sequencing approach provides a 1-step comprehensive molecular analysis of the whole mitochondrial genome for patients in whom a mitochondrial disease is suspected.

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