ABSTRACT
Next‐generation sequencing (NGS) methods are being adopted by genome diagnostics laboratories worldwide. However, implementing NGS‐based tests according to diagnostic standards is a ...challenge for individual laboratories. To facilitate the implementation of NGS in Dutch laboratories, the Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL) set up a working group in 2012. The results of their discussions are presented here. We provide best practice guidelines and criteria for implementing and validating NGS applications in a clinical setting. We introduce the concept of “diagnostic yield” as the main performance characteristic for evaluating diagnostic tests. We recommend that the laboratory procedures, including the tested genes, should be recorded in a publicly available document describing the complete “diagnostic routing.” We also propose that laboratories should use a list of “core disease genes” for specific genetic diseases. This core list contains the essential genes for each disease, and they should all be included in a diagnostic test to establish a reliable and accurate molecular diagnosis. The guidelines will ensure a clear and standardized quality of care provided by genetic diagnostic laboratories. The best practice guidelines and criteria that are presented here were adopted by the VKGL in January 2013.
Abstract Background Aneurysms affecting the aorta are a common condition associated with high mortality as a result of aortic dissection or rupture. Investigations of the pathogenic mechanisms ...involved in syndromic types of thoracic aortic aneurysms, such as Marfan and Loeys-Dietz syndromes, have revealed an important contribution of disturbed transforming growth factor (TGF)-β signaling. Objectives This study sought to discover a novel gene causing syndromic aortic aneurysms in order to unravel the underlying pathogenesis. Methods We combined genome-wide linkage analysis, exome sequencing, and candidate gene Sanger sequencing in a total of 470 index cases with thoracic aortic aneurysms. Extensive cardiological examination, including physical examination, electrocardiography, and transthoracic echocardiography was performed. In adults, imaging of the entire aorta using computed tomography or magnetic resonance imaging was done. Results Here, we report on 43 patients from 11 families with syndromic presentations of aortic aneurysms caused by TGFB3 mutations. We demonstrate that TGFB3 mutations are associated with significant cardiovascular involvement, including thoracic/abdominal aortic aneurysm and dissection, and mitral valve disease. Other systemic features overlap clinically with Loeys-Dietz, Shprintzen-Goldberg, and Marfan syndromes, including cleft palate, bifid uvula, skeletal overgrowth, cervical spine instability and clubfoot deformity. In line with previous observations in aortic wall tissues of patients with mutations in effectors of TGF-β signaling ( TGFBR1/2, SMAD3, and TGFB2 ), we confirm a paradoxical up-regulation of both canonical and noncanonical TGF-β signaling in association with up-regulation of the expression of TGF-β ligands. Conclusions Our findings emphasize the broad clinical variability associated with TGFB3 mutations and highlight the importance of early recognition of the disease because of high cardiovascular risk.
DNA double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the ...NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends were in dynamic equilibrium with Ku70/80 in solution, showing that NHEJ complex assembly is reversible. Accumulation of XRCC4/ligase IV on DSBs depended on the presence of Ku70/80, but not$DNA-PK_{cs}$. We detected a direct interaction between Ku70 and XRCC4 that could explain these requirements. Our results suggest that this assembly constitutes the core of the NHEJ reaction and that XRCC4 may serve as a flexible tether between Ku70/80 and ligase IV.
Although studies on uveal melanoma (UM) revealed prognostic significance of chromosomal aberrations, they resulted in classification errors in survival prediction. A robust prognostic classifier with ...strong predictive value and further insight in genes responsible for poor prognosis were obtained by performing a gene-expression profile in tumors of UM patients for which extensive clinical, histopathologic, cytogenetic, and follow-up data were available. Furthermore, the UM microarray expression data were compared with cytogenetic data.
Gene-expression profiles of 46 UMs were obtained with microchip assays. Data were analyzed with cluster-analysis and predictive analysis of microarrays (PAM) software and validated with real-time PCR. The prognostic significance of UMs with specific molecular signatures was determined. Furthermore, LAP analysis resulted in the identification of differentially expressed chromosomal regions.
The primary UMs were classified in two distinct molecular classes with a strong prognostic value (P < 0.001; hazard ratio 7.7). Classifier gene sets for microarray class and disease-free survival were validated with real-time PCR, and the predictive value of the UM class marker set was validated with gene-expression profiles of tumors provided by other institutions, showing a sensitivity of 0.93 and specificity of 1.00 for class II tumors. A locally adaptive statistical procedure identified two regions on the short arm of chromosome 3 with decreased gene-expression in tumors with shorter disease-free survival.
Microarray classification outperforms known prognostic indicators for UM, such as clinical, histopathologic, and cytogenetic parameters. In addition, the identified regions with lower expressed genes on 3p could harbor genes that are responsible for the poor prognosis of patients with UM.
Objective
Mucopolysaccharidosis (MPS) IIIA (Sanfilippo syndrome type A) is a lysosomal storage disorder caused by deficiency of the enzyme sulfamidase. Information on the natural course of MPS IIIA ...is scarce, but is much needed in view of emerging therapies.
Methods
Clinical history and molecular defects of all 110 MPS IIIA patients identified by enzymatic studies in the Netherlands were collected and included in this study.
Results
First clinical signs, mainly consisting of delayed speech development and behavioral problems, were noted between the ages of 1 and 6 years. Other symptoms included sleeping and hearing problems, recurrent upper airway infections, diarrhea, and epilepsy. The clinical course varied remarkably and could be correlated with the molecular defects. The frequent pathogenic mutations p.R245H, p.Q380R, p.S66W, and c.1080delC were associated with the classical severe phenotype. Patients compound heterozygous for the p.S298P mutation in combination with 1 of the mutations associated with the classical severe phenotype had a significantly longer preservation of psychomotor functions and a longer survival. Two patients homozygous for the p.S298P mutation, and 4 patients from 3 families heterozygous for 3 missense variants not reported previously (p.T421R, p.P180L, and p.L12Q), showed a remarkably attenuated phenotype.
Interpretation
We report the natural history and mutational analysis in a large unbiased cohort of MPS IIIA patients. We demonstrate that the clinical spectrum of MPS IIIA is much broader than previously reported. A significant genotype‐phenotype correlation was established in this cohort. Ann Neurol 2010
Exome sequencing (ES) enhanced the diagnostic yield of genetic testing, but has also increased the possibility of uncertain findings. Prenatal ES is increasingly being offered after a fetal ...abnormality is detected through ultrasound. It is important to know how to handle uncertainty in this particularly stressful period. This systematic review aimed to provide a comprehensive overview of guidelines available for addressing uncertainty related to prenatal chromosomal microarray (CMA) and ES. Ten uncertainty types associated with prenatal ES and CMA were identified and defined by an international multidisciplinary team. Medline (all) and Embase were systematically searched. Laboratory scientists, clinical geneticists, psychologists, and a fetal medicine specialist screened the papers and performed the data extraction. Nineteen papers were included. Recommendations generally emphasized the importance of trio analysis, clinical information, data sharing, validation and re‐analysis, protocols, multidisciplinary teams, genetic counselling, whether to limit the possible scope of results, and when to report particular findings. This systematic review helps provide a vocabulary for uncertainties, and a compass to navigate uncertainties. Prenatal CMA and ES guidelines provide a strong starting point for determining how to handle uncertainty. Gaps in guidelines and recommendations were identified and discussed to provide direction for future research and policy making.
Guidelines for prenatal chromosomal microarray (CMA) and Exome Sequencing (ES) were identified through a systematic review and summarized within a framework of ten defined uncertainty types associated with this prenatal genetic testing. This paper aimed to provide a vocabulary for prenatal genetic uncertainty, as well as a compass to navigate these uncertainties.
Objective
Individuals with 45,X/46,XY or 46,XY gonadal dysgenesis are at increased risk of germ cell malignancies. Therefore, prophylactic bilateral gonadectomy is advised in girls and considered in ...boys with atypical genitalia for undescended, macroscopically abnormal gonads. However, severely dysgenetic gonads may not contain germ cells rendering gonadectomy unnecessary. Therefore, we investigate if undetectable preoperative serum anti‐Müllerian hormone (AMH) and inhibin B can predict the absence of germ cells, (pre)malignant or otherwise.
Design, Patients and Measurements
Individuals who had undergone bilateral gonadal biopsy and/or gonadectomy because of suspected gonadal dysgenesis in 1999–2019 were included in this retrospective study if preoperative AMH and/or inhibin B were available. Histological material was reviewed by an experienced pathologist. Haematoxylin and eosin and immunohistochemical stainings for SOX9, OCT4, TSPY and SCF (KITL) were used.
Results
Thirteen males and 16 females were included, 20 with 46,XY and 9 with 45,X/46,XY DSD. Three females had dysgerminoma alongside gonadoblastoma; two gonadoblastoma, one germ cell neoplasia in situ (GCNIS) and three males had pre‐GCNIS and/or pre‐gonadoblastoma. Gonadoblastoma and/or dysgerminoma were present in 3/11 individuals with undetectable AMH and inhibin B, one of whom also had non‐(pre)malignant germ cells. Of the other 18, in whom AMH and/or inhibin B were detectable, only one had no germ cells.
Conclusions
Undetectable serum AMH and inhibin B cannot reliably predict the absence of germ cells and germ cell tumours in individuals with 45,X/46,XY or 46,XY gonadal dysgenesis. This information should help in counselling about prophylactic gonadectomy, taking into account both the germ cell cancer risk and potential for gonadal function.
To investigate the presence of focal or diffuse heterogeneity of monosomy 3 in uveal melanoma, by using fluorescence in situ hybridization (FISH).
Direct interphase FISH in a series of 151 uveal ...melanomas revealed 82 tumors with loss of chromosome 3. Tumors with monosomy 3 were suspected to be heterogeneous if there were low percentages of monosomy 3, triploid clones, inconsistencies between FISH on centromere 3 and the long arm of chromosome 3, or discrepancies between fine-needle-aspiration biopsies (FNABs) and the main tumor. These tumors (n=16), all choroidal melanomas, were selected and analyzed for intratumor heterogeneity by using FISH on paraffin-embedded tissue sections.
Different sections of each tumor were evaluated with FISH: 6 tumors showed monosomy 3 in the same percentage throughout the tumor, and 10 showed multiple clones with different percentages of monosomy 3. However, these tumors did not show focal heterogeneity with respect to chromosome 3 status, and differences in monosomy 3 distribution between the base and apex of the tumor could not be identified.
Although a small number of uveal melanomas show heterogeneity for chromosome 3, it does not affect survival. In the presence of triploid clones, the loss of chromosome 3 is more difficult to interpret. In general, tumor biopsies in uveal melanoma provide an accurate prediction of the patient's prognosis.
We describe monozygotic twin girls with genetic variation at two separate loci resulting in a blended phenotype of Prader–Willi syndrome and Pitt–Hopkins syndrome. These girls were diagnosed in early ...infancy with Prader–Willi syndrome, but developed an atypical phenotype, with apparent intellectual deficiency and lack of obesity. Array‐comparative genomic hybridization confirmed a de novo paternal deletion of the 15q11.2q13 region and exome sequencing identified a second mutational event in both girls, which was a novel variant c.145+1G>A affecting a TCF4 canonical splicing site inherited from the mosaic mother. RNA studies showed that the variant abolished the donor splicing site, which was accompanied by activation of an alternative non‐canonical splicing‐site which then predicts a premature stop codon in the following exon. Clinical re‐evaluation of the twins indicated that both variants are likely contributing to the more severe phenotypic presentation. Our data show that atypical clinical presentations may actually be the expression of blended clinical phenotypes arising from independent pathogenic events at two loci.
PDF
