Epigenetics is currently in an exponential phase of growth, constituting one of the most promising fields in science, particularly in cancer research. Impaired epigenetic processes can lead to ...abnormal gene activity or inactivity, causing cellular disorders that are closely associated with tumor initiation and progression. Thus, there is a pivotal role of massive sequencing techniques for epigenetics, which aim to find novel biomarkers, factors of prognosis and prediction, and targets for achieving personalized treatments. We present a brief description of the evolution of next-generation sequencing technologies and its coupling with DNA methylation analysis techniques, highlighting its future in translational medicine and presenting significant findings in several malignancies. We also expose critical topics related to the implementation of these approaches, which is expected to be affordable for most research centers in the near future.
Genetic analysis using a next-generation sequencing (NGS)–customized panel allowed us to exclude mutations in all previously reported PID-related genes. Because of the complexity of the clinical ...manifestations, a single nucleotide polymorphism (SNP) array was performed with DNA from the patient and her parents. At the age of 20 months, after PID confirmation, allogeneic hematopoietic stem cell transplantation was proposed to control infectious risk, eosinophilic/lymphocytic tissue damage, and lymphoproliferation. Because of the lack of a matched related or unrelated donor, which was unanticipated given the chromosome 6 homozygosity that affected the HLA loci, a haploidentical donor was considered. ...Treg cells were in the low-to-normal range (see Table E3). Because of the uncertain long-term immunologic and clinical consequences of this novel mutation in heterozygous individuals, the maternal grandfather was considered a more suitable haploidentical donor in this complex clinical situation. According to best practices proposed by GATK, indel realignment was done to determine suspicious intervals likely to need realignment (GATK RealignerTargetCreator function), followed by local realignment of reads to correct misalignments caused by indels (GATK IndelRealigner function).
Early-onset high myopia (EoHM) is a disease that causes a spherical refraction error of ≥-6 diopters before 10 years of age, with potential multiple ocular complications. In this article, we report a ...clinical and genetic study of 43 families with EoHM recruited in our center. A complete ophthalmological evaluation was performed, and a sample of peripheral blood was obtained from proband and family members. DNA was analyzed using a customized next-generation sequencing panel that included 419 genes related to ophthalmological disorders with a suspected genetic cause, and genes related to EoHM pathogenesis. We detected pathogenic and likely pathogenic variants in 23.9% of the families and detected variants of unknown significance in 76.1%. Of these, 5.7% were found in genes related to non-syndromic EoHM, 48.6% in genes associated with inherited retinal dystrophies that can include a syndromic phenotype, and 45.7% in genes that are not directly related to EoHM or retinal dystrophy. We found no candidate genes in 23% of the patients, which suggests that further studies are needed. We propose a systematic genetic analysis for patients with EoHM because it helps with follow-up, prognosis and genetic counseling.
Chromosomal deletions at 11p13 are a frequent cause of congenital Aniridia, a rare pan-ocular genetic disease, and of WAGR syndrome, accounting up to 30% of cases. First-tier genetic testing for ...newborn with aniridia, to detect 11p13 rearrangements, includes Multiplex Ligation-dependent Probe Amplification (MLPA) and karyotyping. However, neither of these approaches allow obtaining a complete picture of the high complexity of chromosomal deletions and breakpoints in aniridia. Here, we report the development and validation of a customized targeted array-based comparative genomic hybridization, so called WAGR-array, for comprehensive high-resolution analysis of CNV in the WAGR locus. Our approach increased the detection rate in a Spanish cohort of 38 patients with aniridia, WAGR syndrome and other related ocular malformations, allowing to characterize four undiagnosed aniridia cases, and to confirm MLPA findings in four additional patients. For all patients, breakpoints were accurately established and a contiguous deletion syndrome, involving a large number of genes, was identified in three patients. Moreover, we identified novel microdeletions affecting 3' PAX6 regulatory regions in three families with isolated aniridia. This tool represents a good strategy for the genetic diagnosis of aniridia and associated syndromes, allowing for a more accurate CNVs detection, as well as a better delineation of breakpoints. Our results underline the clinical importance of performing exhaustive and accurate analysis of chromosomal rearrangements for patients with aniridia, especially newborns and those without defects in PAX6 after diagnostic screening.
Our purpose was to identify mutations responsible for non-syndromic congenital cataracts through the implementation of next-generation sequencing (NGS) in our center. A sample of peripheral blood was ...obtained from probands and willing family members and genomic DNA was extracted from leukocytes. DNA was analyzed implementing a panel (OFTv2.1) including 39 known congenital cataracts disease genes. 62 probands from 51 families were recruited. Pathogenic or likely pathogenic variants were identified in 32 patients and 25 families; in 16 families (64%) these were de novo mutations. The mutation detection rate was 49%. Almost all reported mutations were autosomal dominant. Mutations in crystallin genes were found in 30% of the probands. Mutations in membrane proteins were detected in seven families (two in
and five in
). Mutations in
and
were each found in three families. Other mutations detected affected
and
. Variants classified as of unknown significance were found in 5 families (9.8%), affecting
and
. Mutations lead to different cataract phenotypes within the same family.
Several new microdeletion and microduplication syndromes are emerging as disorders that have been proven to cause multisystem pathologies frequently associated with intellectual disability (ID), ...multiple congenital anomalies (MCA), autistic spectrum disorders (ASD) and other phenotypic findings. In this paper, we review the "new" and emergent microdeletion and microduplication syndromes that have been described and recognized in recent years with the aim of summarizing their main characteristics and chromosomal regions involved. We decided to group them by genomic region and within these groupings have classified them into those that include ID, MCA, ASD or other findings. This review does not intend to be exhaustive but is rather a quick guide to help pediatricians, clinical geneticists, cytogeneticists and/or molecular geneticists.
A clinical and genetic study was conducted with pediatric patients and their relatives with optic atrophy 1 (
mutations to establish whether there is a genotype-phenotype correlation among the ...variants detected within and between families. Eleven children with a confirmed
mutation were identified during the study period. The main initial complaint was reduced visual acuity (VA), present in eight patients of the cohort. Eight of eleven patients had a positive family history of optic atrophy. The mean visual acuity at the start of the study was 0.40 and 0.44 LogMAR in the right and left eye, respectively. At the end of the study, the mean visual acuity was unchanged. Optical coherence tomography during the first visit showed a mean retinal nerve fiber layer thickness of 81.6 microns and 80.5 microns in the right and left eye, respectively; a mean ganglion cell layer of 52.5 and 52.4 microns, respectively, and a mean central macular thickness of 229.5 and 233.5 microns, respectively. The most common visual field defect was a centrocecal scotoma, and nine out of eleven patients showed bilateral temporal disc pallor at baseline. Sequencing of
showed seven different mutations in the eleven patients, one of which, NM_130837.3: c.1406_1407del (p.Thr469LysfsTer16), has not been previously reported. Early diagnosis of dominant optic atrophy is crucial, both for avoiding unnecessary consultations and/or treatments and for appropriate genetic counseling.
CRB1 mutations are reported as cause of severe congenital and early-onset retinal dystrophies (EORD) with different phenotypic manifestations, including Leber congenital amaurosis (LCA), retinitis ...pigmentosa (RP) and cone-rod dystrophies. Comprehensive mutational scanning of the whole gene has been only performed in few cohorts, mainly in LCA patients. Here, we aimed investigating the real prevalence of CRB1 mutations in the Spanish population by extensive screening of CRB1 mutations in a large cohort of LCA and EORP cases.
This report integrates data from previous studies on CRB1 defects in our Spanish cohort of LCA and early-onset RP (EORP) with new findings from a comprehensive mutational screening of the whole gene. The molecular tools used include mutation genotyping arrays, whole-genome homozygosity mapping, an optimized high-resolution melting (HRM) analysis and Sanger sequencing.
A large clinically well-characterized cohort of 404 Spanish cases was studied, 114 of which suffered from LCA and 290 from EORP. This study reveals that 11% of Spanish patients carried mutations in CRB1, ranging from 9% of EORP to 14% of LCA cases. More than three quarters of the mutations identified herein have been first described in this Spanish cohort, 13 of them are unreported new variants and 13 had been previously reported in our previous studies.
This work provides a wide spectrum of CRB1 mutations in the Spanish EORD patients and evidences the major role of CRB1 as causal gene in the Spanish EORP patients. It is noteworthy that a high rate of private mutations only described in our cohort has been found so far. To our knowledge, this study represents the most complete mutational screening of CRB1 in a Spanish LCA and EORP cohort, allowing us to establish gene-specific frequencies and to provide a wide spectrum of CRB1 mutations in the Spanish population.
Wilms tumor (WT), the most common cancer of the kidney in infants and children, has a complex etiology that is still poorly understood. Identification of genomic copy number variants (CNV) in tumor ...genomes provides a better understanding of cancer development which may be useful for diagnosis and therapeutic targets. In paired blood and tumor DNA samples from 14 patients with sporadic WT, analyzed by aCGH, 22% of chromosome abnormalities were novel. All constitutional alterations identified in blood were segmental (in 28.6% of patients) and were also present in the paired tumor samples. Two segmental gains (2p21 and 20q13.3) and one loss (19q13.31) present in blood had not been previously described in WT. We also describe, for the first time, a small, constitutive partial gain of 3p22.1 comprising 2 exons of CTNNB1, a gene associated to WT. Among somatic alterations, novel structural chromosomal abnormalities were found, like gain of 19p13.3 and 20p12.3, and losses of 2p16.1-p15, 4q32.5-q35.1, 4q35.2-q28.1 and 19p13.3. Candidate genes included in these regions might be constitutively (SIX3, SALL4) or somatically (NEK1, PIAS4, BMP2) operational in the development and progression of WT. To our knowledge this is the first report of CNV in paired blood and tumor samples in sporadic WT.
Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct ...somatic
variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same
variants occur in
-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of
(His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating
mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.
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