DLG4-related synaptopathy: a new rare brain disorder.

PURPOSE: Postsynaptic density protein-95 (PSD-95), encoded by DLG4, regulates excitatory synaptic function in the brain. Here we present the clinical and genetic features of 53 patients (42 previously unpublished) with DLG4 variants. METHODS: The clinical and genetic information were collected through GeneMatcher collaboration. All the individuals were investigated by local clinicians and the gene variants were identified by clinical exome/genome sequencing. RESULTS: The clinical picture was predominated by early onset global developmental delay, intellectual disability, autism spectrum disorder, and attention deficit-hyperactivity disorder, all of which point to a brain disorder. Marfanoid habitus, which was previously suggested to be a characteristic feature of DLG4-related phenotypes, was found in only nine individuals and despite some overlapping features, a distinct facial dysmorphism could not be established. Of the 45 different DLG4 variants, 39 were predicted to lead to loss of protein function and the majority occurred de novo (four with unknown origin). The six missense variants identified were suggested to lead to structural or functional changes by protein modeling studies. CONCLUSION: The present study shows that clinical manifestations associated with DLG4 overlap with those found in other neurodevelopmental disorders of synaptic dysfunction; thus, we designate this group of disorders as DLG4-related synaptopathy.

Putting genome-wide sequencing in neonates into perspective.

PURPOSE: Several studies have reported diagnostic yields up to 57% for rapid exome or genome sequencing (rES/GS) as a single test in neonatal intensive care unit (NICU) patients, but the additional yield of rES/GS compared with other available diagnostic options still remains unquantified in this population. METHODS: We retrospectively evaluated all genetic NICU consultations in a 2-year period. RESULTS: In 132 retrospectively evaluated NICU consultations 27 of 32 diagnoses (84.4%) were made using standard genetic workup. Most diagnoses (65.6%) were made within 16 days. Diagnostic ES yield was 5/29 (17.2%). Genetic diagnoses had a direct effect on clinical management in 90.6% (29/32) of patients. CONCLUSIONS: Our study shows that exome sequencing has a place in NICU diagnostics, but given the associated costs and the high yield of alternative diagnostic strategies, we recommend to first perform clinical genetic consultation.

Correction: Putting genome-wide sequencing in neonates into perspective.

The original version of this Article contained an error in the spelling of the author Pleuntje J. van der Sluijs, which was incorrectly given as Eline (P. J.) van der Sluijs. This has now been corrected in both the PDF and HTML versions of the Article.

The BRCA1 c. 5096G>A p.Arg1699Gln (R1699Q) intermediate risk variant: breast and ovarian cancer risk estimation and recommendations for clinical management from the ENIGMA consortium.

BACKGROUND: We previously showed that the BRCA1 variant c.5096G>A p.Arg1699Gln (R1699Q) was associated with an intermediate risk of breast cancer (BC) and ovarian cancer (OC). This study aimed to assess these cancer risks for R1699Q carriers in a larger cohort, including follow-up of previously studied families, to further define cancer risks and to propose adjusted clinical management of female BRCA1*R1699Q carriers. METHODS: Data were collected from 129 BRCA1*R1699Q families ascertained internationally by ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium members. A modified segregation analysis was used to calculate BC and OC risks. Relative risks were calculated under both monogenic model and major gene plus polygenic model assumptions. RESULTS: In this cohort the cumulative risk of BC and OC by age 70 years was 20% and 6%, respectively. The relative risk for developing cancer was higher when using a model that included the effects of both the R1699Q variant and a residual polygenic component compared with monogenic model (for BC 3.67 vs 2.83, and for OC 6.41 vs 5.83). CONCLUSION: Our results confirm that BRCA1*R1699Q confers an intermediate risk for BC and OC. Breast surveillance for female carriers based on mammogram annually from age 40 is advised. Bilateral salpingo-oophorectomy should be considered based on family history.

Substantial evidence for the clinical significance of missense variant BRCA1 c.5309G>T p.(Gly1770Val).

PURPOSE: Classification of rare BRCA1 missense variants presents a major challenge for the counseling and treatment of patients. Variant classification can be complicated by conflicting lines of evidence. BRCA1 c.5309G>T p.(Gly1770Val) has been shown to abrogate BRCA1 protein homologous DNA repair; however, multiple sequence alignment demonstrates a lack of sequence conservation at this position, suggesting that glycine at position 1770 may not be essential for cellular maintenance in humans. We analyzed clinical information to resolve the classification of BRCA1 c.5309G>T p.(Gly1770Val). METHODS: We performed multifactorial likelihood analysis combining segregation data for 14 informative families, and breast tumor histopathological data for 17 variant carriers, ascertained through the ENIGMA consortium. RESULTS: Bayes segregation analysis gave a likelihood ratio of 101:1 in favor of pathogenicity. The vast majority of breast tumors showed features indicative of pathogenic variant carrier status, resulting in a likelihood ratio of 15800794:1 towards pathogenicity. Despite a low prior probability of pathogenicity (0.03) based on bioinformatic prediction, multifactorial likelihood analysis including segregation and histopathology analysis gave a posterior probability of > 0.99 and final classification of Pathogenic. CONCLUSIONS: We provide evidence that BRCA1 c.5309G>T p.(Gly1770Val), previously described as a Moroccan founder variant, should be treated as a disease-causing variant despite a lack of evolutionary conservation at this amino acid position. Additionally, we stress that bioinformatic information should be used in combination with other data, either direct clinical evidence or some form of clinical calibration, to arrive at a final clinical classification.

Classification and Clinical Management of Variants of Uncertain Significance in High Penetrance Cancer Predisposition Genes.

In 2008, the International Agency for Research on Cancer (IARC) proposed a system for classifying sequence variants in highly penetrant breast and colon cancer susceptibility genes, linked to clinical actions. This system uses a multifactorial likelihood model to calculate the posterior probability that an altered DNA sequence is pathogenic. Variants between 5%-94.9% (class 3) are categorized as variants of uncertain significance (VUS). This interval is wide and might include variants with a substantial difference in pathogenicity at either end of the spectrum. We think that carriers of class 3 variants would benefit from a fine-tuning of this classification. Classification of VUS to a category with a defined clinical significance is very important because for carriers of a pathogenic mutation full surveillance and risk-reducing surgery can reduce cancer incidence. Counselees who are not carriers of a pathogenic mutation can be discharged from intensive follow-up and avoid unnecessary risk-reducing surgery. By means of examples, we show how, in selected cases, additional data can lead to reclassification of some variants to a different class with different recommendations for surveillance and therapy. To improve the clinical utility of this classification system, we suggest a pragmatic adaptation to clinical practice.

A terminal 3p26.3 deletion is not associated with dysmorphic features and intellectual disability in a four-generation family.

Terminal deletions of the distal part of the short arm of chromosome 3 cause a wide range of phenotypes from normal to dysmorphic including microcephaly, developmental delay and intellectual disability. We studied the clinical consequences of a terminal deletion of the short arm of chromosome 3 in four generations of a family. The index patient is a14-month-old boy with microcephaly, corpus callosum dysgenesis, and minor dysmorphic features. Single Nucleotide Polymorphism (SNP) array analysis detected a duplication on the long arm of chromosome 6. His apparently healthy mother carries the same 6q duplication, but as an unexpected finding a terminal deletion of 2.9 Mb of the short arm of chromosome 3 was observed. Further co-segregation analysis in the family for the chromosome 3 deletion showed that with the exception of the sister of the index who has autism, speech delay, and learning problems, family members in four generations of this family are carrier of this 3p deletion and apparently healthy. To our knowledge, this is the first report of a study of this terminal 3p deletion in four generations. In this report, we review the literature on terminal 3p deletions and discuss the importance of molecular testing and reporting of copy number variants to achieve accurate genetic counseling in prenatal and postnatal screening.

Variants of uncertain significance in BRCA1 and BRCA2 assessment of in silico analysis and a proposal for communication in genetic counselling.

BACKGROUND: Nearly 15% of BRCA1 and BRCA2 DNA tests lead to the identification of Variants of Uncertain Significance (VUS). VUS are classified in the Netherlands according to the Bell system and it is current practice that class III VUS are communicated to counsellees, but not class II or lower VUS. Our aims were to investigate the utility of in silico characteristics in the classification of VUS and whether initial VUS classifications justify differences in communication protocols during counselling. METHODS: We classified 88 missense VUS in BRCA1 and BRCA2 on the basis of an in silico analysis and compared the classification of a subset of 60 VUS of which additional information including family, genetic and tumour data was available. RESULTS: VUS allocated to class III more frequently showed in silico indications of a deleterious effect than class II VUS. Of the 46 VUS assigned to class II by in silico analysis alone, nearly half were eventually recategorised as class I and 10% as class III when additional information was included. CONCLUSIONS: As in silico analysis alone is not always sufficient to unambiguously assign VUS to either class II or class III, we would argue that the prospect of obtaining additional information from a family should be given more weight during the decision process preceding the communication of a VUS test result. Research initiatives such as the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA), which strive to combine diverse sources of information, will be valuable in aiding a definitive classification of a VUS.

Case series, chemotherapy-induced cardiomyopathy: mind the family history!

BACKGROUND: Cardiotoxicity presenting as cardiomyopathy is a common side effect in cancer treatment especially with anthracyclines. The role of genetic predisposition is still being investigated. CASE SUMMARY: Four unrelated patients with a familial burden for cardiac disease, who developed cardiomyopathy after anthracycline treatment are presented. Case 1 received chemotherapy for breast cancer and developed a dilated left ventricle just after treatment. Her father had died unexpectedly while being screened for heart transplant. Case 2 was known with a family history of sudden cardiac death prior to her breast cancer diagnosis. She received anthracycline-containing chemotherapy treatment twice in 5 years due to recurrence of breast cancer. During that period, two brothers developed a cardiomyopathy. Eighteen years later, a genetic predisposition for cardiomyopathy was ascertained and at screening an asymptomatic non-ischaemic cardiomyopathy was established. Case 3 was diagnosed with a dilated cardiomyopathy 1 year after chemotherapy treatment for breast cancer. Her mother had developed a dilated cardiomyopathy several years before. Case 4 received chemotherapy treatment for Non-Hodgkin's lymphoma and developed dilated cardiomyopathy 1 year later. His brother died from congestive heart failure which he developed after chemotherapy for Non-Hodgkin's lymphoma and a grandmother had died suddenly during child delivery. In all four cases, genetic screening showed (likely) pathogenic variants in cardiomyopathy-associated genes. DISCUSSION: Current guidelines recommend cardiac evaluation in cancer patients receiving chemotherapy based on the presence of cardiovascular risk factors at the start of treatment. This series emphasizes the importance of including a thorough family history in this process.

Functional Categorization of BRCA1 Variants of Uncertain Clinical Significance in Homologous Recombination Repair Complementation Assays.

PURPOSE: Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway. EXPERIMENTAL DESIGN: Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1-deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families. RESULTS: All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants. CONCLUSIONS: We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.

Genetic Testing and Clinical Management Practices for Variants in Non-BRCA1/2 Breast (and Breast/Ovarian) Cancer Susceptibility Genes: An International Survey by the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) Clinical Working Group.

PURPOSE: To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non-BRCA1/2 genes. METHODS: Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy. RESULTS: Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six (PALB2, TP53, PTEN, CHEK2, ATM, and BRIP1) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53, PTEN, and other cancer syndrome-associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations. CONCLUSION: Currently, a small number of genes beyond BRCA1/2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

Downregulation of vertebrate Tel (ETV6) and Drosophila Yan is facilitated by an evolutionarily conserved mechanism of F-box-mediated ubiquitination.

The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue, Yan, are both indispensable for development, and they orchestrate cell growth and differentiation by binding to DNA, thus inhibiting gene expression. To trigger cell differentiation, these barriers to transcriptional activation must be relieved, and it is established that posttranslational modifications, such as phosphorylation and sumoylation, can specifically impair the repressive functions of Tel and Yan and are crucial for modulating their transcriptional activity. To date, however, relatively little is known about the control of Tel and Yan protein degradation. In recent years, there has been a concentrated effort to assign functions to the large number of F-box proteins encoded by both vertebrate and invertebrate genomes. Here, we report the identification and characterization of a previously unreported, evolutionarily conserved F-box protein named Fbl6. We isolated both human and Drosophila melanogaster fbl6 cDNA and show that the encoded Fbl6 protein binds to both Tel and Yan via their SAM domains. We demonstrate that both Tel and Yan are ubiquitinated, a process which is stimulated by Fbl6 and leads to proteasomal degradation. We recently established that the sumoylation of Tel on lysine 11 negatively regulates its repressive function and that the sumoylation of Tel monomers, but not that of Tel oligomers, may sensitize Tel for proteasomal degradation. Here, we found that Fbl6 regulates Tel/Yan protein stability and allows appropriate spatiotemporal control of gene expression by these repressors.