Developmental epileptic encephalopathy in DLG4-related synaptopathy.

OBJECTIVE: The postsynaptic density protein of excitatory neurons PSD-95 is encoded by discs large MAGUK scaffold protein 4 (DLG4), de novo pathogenic variants of which lead to DLG4-related synaptopathy. The major clinical features are developmental delay, intellectual disability (ID), hypotonia, sleep disturbances, movement disorders, and epilepsy. Even though epilepsy is present in 50% of the individuals, it has not been investigated in detail. We describe here the phenotypic spectrum of epilepsy and associated comorbidities in patients with DLG4-related synaptopathy. METHODS: We included 35 individuals with a DLG4 variant and epilepsy as part of a multicenter study. The DLG4 variants were detected by the referring laboratories. The degree of ID, hypotonia, developmental delay, and motor disturbances were evaluated by the referring clinician. Data on awake and sleep electroencephalography (EEG) and/or video-polygraphy and brain magnetic resonance imaging were collected. Antiseizure medication response was retrospectively assessed by the referring clinician. RESULTS: A large variety of seizure types was reported, although focal seizures were the most common. Encephalopathy related to status epilepticus during slow-wave sleep (ESES)/developmental epileptic encephalopathy with spike-wave activation during sleep (DEE-SWAS) was diagnosed in >25% of the individuals. All but one individual presented with neurodevelopmental delay. Regression in verbal and/or motor domains was observed in all individuals who suffered from ESES/DEE-SWAS, as well as some who did not. We could not identify a clear genotype-phenotype relationship even between individuals with the same DLG4 variants. SIGNIFICANCE: Our study shows that a subgroup of individuals with DLG4-related synaptopathy have DEE, and approximately one fourth of them have ESES/DEE-SWAS. Our study confirms DEE as part of the DLG4-related phenotypic spectrum. Occurrence of ESES/DEE-SWAS in DLG4-related synaptopathy requires proper investigation with sleep EEG.

Determinants Affecting the Clinical Implementation of a Molecularly Informed Molecular Tumor Board Recommendation: Experience from a Tertiary Cancer Center.

Molecular Tumor Boards (MTBs) converge state-of-the-art next-generation sequencing (NGS) methods with the expertise of an interdisciplinary team consisting of clinicians, pathologists, human geneticists, and molecular biologists to provide molecularly informed guidance in clinical decision making to the treating physician. In the present study, we particularly focused on elucidating the factors impacting on the clinical translation of MTB recommendations, utilizing data generated from gene panel mediated comprehensive genomic profiling (CGP) of 554 patients at the MTB of the Comprehensive Cancer Center Erlangen, Germany, during the years 2016 to 2020. A subgroup analysis of cases with available follow-up data (n = 332) revealed 139 cases with a molecularly informed MTB recommendation, which was successfully implemented in the clinic in 44 (31.7%) of these cases. Here, the molecularly matched treatment was applied in 45.4% (n = 20/44) of cases for ≥6 months and in 25% (n = 11/44) of cases for 12 months or longer (median time to treatment failure, TTF: 5 months, min: 1 month, max: 38 months, ongoing at data cut-off). In general, recommendations were preferentially implemented in the clinic when of high (i.e., tier 1) clinical evidence level. In particular, this was the case for MTB recommendations suggesting the application of PARP, PIK3CA, and IDH1/2 inhibitors. The main reason for non-compliance to the MTB recommendation was either the application of non-matched treatment modalities (n = 30)/stable disease (n = 7), or deteriorating patient condition (n = 22)/death of patient (n = 9). In summary, this study provides an insight into the factors affecting the clinical implementation of molecularly informed MTB recommendations, and careful considerations of these factors may guide future processes of clinical decision making.

Ex Vivo Chromosomal Radiosensitivity Testing in Patients with Pathological Germline Variants in Breast Cancer High-Susceptibility Genes BReast CAncer 1 and BReast CAncer 2.

BACKGROUND: Individual radiosensitivity is an important factor in the occurrence of undesirable consequences of radiotherapy. The potential for increased radiosensitivity has been linked to highly penetrant heterozygous mutations in DNA repair genes such as BRCA1 and BRCA2. By studying the chromosomal radiosensitivity of BRCA1/2 mutation carriers compared to the general population, we study whether increased chromosomal radiation sensitivity is observed in patients with BRCA1/2 variants. METHODS: Three-color-fluorescence in situ hybridization was performed on ex vivo-irradiated peripheral blood lymphocytes from 64 female patients with a heterozygous germline BRCA1 or BRCA2 mutation. Aberrations in chromosomes #1, #2 and #4 were analyzed. Mean breaks per metaphase (B/M) served as the parameter for chromosomal radiosensitivity. The results were compared with chromosomal radiosensitivity in a cohort of generally healthy individuals and patients with rectal cancer or breast cancer. RESULTS: Patients with BRCA1/2 mutations (n = 64; B/M 0.47) overall showed a significantly higher chromosomal radiosensitivity than general healthy individuals (n = 211; B/M 0.41) and patients with rectal cancer (n = 379; B/M 0.44) and breast cancer (n = 147; B/M 0.45) without proven germline mutations. Chromosomal radiosensitivity varied depending on the locus of the BRCA1/2 mutation. CONCLUSIONS: BRCA1/2 mutations result in slightly increased chromosomal sensitivity to radiation. A few individual patients have a marked increase in radiation sensitivity. Therefore, these patients are at a higher risk for adverse therapeutic consequences.

Familial acute aortic dissection associated with a novel ACTA2 germline variant.

Aortic dissection is a life-threatening cardiovascular disease. Hereditary disorders are responsible for a small percentage of cases. Nonetheless, it is important to identify genetic causes, as they are often autosomal dominantly inherited and are of life-saving importance if we can identify persons at risk. Mutations of the ACTA2 gene are the most common cause of non-syndromic familial aortic disease. Exploration of the genetic background in suspected familial cases and determination of the exact etiology are mandatory for management and establishing appropriate follow-up strategies due to the risk of fatal recurrences. Herein, we present a 21-year-old male with a familial acute aortic dissection associated with novel ACTA2 germline variant and discuss the management and surveillance considerations.

What's a Biofilm?-How the Choice of the Biofilm Model Impacts the Protein Inventory of Clostridioides difficile.

The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile's V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.

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.

Breast MRI texture analysis for prediction of BRCA-associated genetic risk.

BACKGROUND: BRCA1/2 deleterious variants account for most of the hereditary breast and ovarian cancer cases. Prediction models and guidelines for the assessment of genetic risk rely heavily on criteria with high variability such as family cancer history. Here we investigated the efficacy of MRI (magnetic resonance imaging) texture features as a predictor for BRCA mutation status. METHODS: A total of 41 female breast cancer individuals at high genetic risk, sixteen with a BRCA1/2 pathogenic variant and twenty five controls were included. From each MRI 4225 computer-extracted voxels were analyzed. Non-imaging features including clinical, family cancer history variables and triple negative receptor status (TNBC) were complementarily used. Lasso-principal component regression (L-PCR) analysis was implemented to compare the predictive performance, assessed as area under the curve (AUC), when imaging features were used, and lasso logistic regression or conventional logistic regression for the remaining analyses. RESULTS: Lasso-selected imaging principal components showed the highest predictive value (AUC 0.86), surpassing family cancer history. Clinical variables comprising age at disease onset and bilateral breast cancer yielded a relatively poor AUC (~ 0.56). Combination of imaging with the non-imaging variables led to an improvement of predictive performance in all analyses, with TNBC along with the imaging components yielding the highest AUC (0.94). Replacing family history variables with imaging components yielded an improvement of classification performance of ~ 4%, suggesting that imaging compensates the predictive information arising from family cancer structure. CONCLUSIONS: The L-PCR model uncovered evidence for the utility of MRI texture features in distinguishing between BRCA1/2 positive and negative high-risk breast cancer individuals, which may suggest value to diagnostic routine. Integration of computer-extracted texture analysis from MRI modalities in prediction models and inclusion criteria might play a role in reducing false positives or missed cases especially when established risk variables such as family history are missing.

Targeted sequencing of FH-deficient uterine leiomyomas reveals biallelic inactivating somatic fumarase variants and allows characterization of missense variants.

Uterine leiomyomas (ULs) constitute a considerable health burden in the general female population. The fumarate hydratase (FH) deficient subtype is found in up to 1.6% and can occur in hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome. We sequenced 13 FH deficient ULs from a previous immunohistochemical screen using a targeted panel and identified biallelic FH variants in all. In eight, we found an FH point mutation (two truncating, six missense) with evidence for loss of the second allele. Variant allele-frequencies in all cases with a point mutation pointed to somatic variants. Spatial clustering of the identified missense variants in the lyase domain indicated altered fumarase oligomerization with subsequent degradation as explanation for the observed FH deficiency. Biallelic FH deletions in five tumors confirm the importance of copy number loss as mutational mechanism. By curating all pathogenic FH variants and calculating their population frequency, we estimate a carrier frequency of up to 1/2,563. Comparing with the prevalence of FH deficient ULs, we conclude that most are sporadic and estimate 2.7-13.9% of females with an FH deficient UL to carry a germline FH variant. Further prospective tumor/normal sequencing studies are needed to develop a reliable screening strategy for HLRCC in women with ULs.

Psychiatric Disorders and Distal 21q Deletion-A Case Report.

Partial deletion of chromosome 21q is a very rare genetic condition with highly variable phenotypic features including heart defects, high or cleft palate, brain malformations (e.g., cerebral atrophy), developmental delay and intellectual disability. So far, there is very limited knowledge about psychiatric disorders and their effective treatment in this special population. To fill this gap, the authors present the case of an initially five-year-old girl with distal deletion (del21q22.2) and comorbid oppositional defiant disorder (main psychiatric diagnosis) covering a period of time of almost four years comprising initial psychological/psychiatric assessment, subsequent treatment with Parent-Child Interaction Therapy (PCIT), and follow-up assessments. Post-intervention results including a 19-month follow-up indicated good overall efficacy of PCIT and high parental satisfaction with the treatment. This case report makes a substantial contribution to enhancing knowledge on psychiatric comorbidity and its effective treatment in patients with terminal 21q deletion. Moreover, it emphasizes the necessity of multidisciplinarity in diagnosis and treatment due to the variety of anomalies associated with 21q deletion. Regular screenings for psychiatric disorders and (if indicated) thorough psychological and psychiatric assessment seem to be reasonable in most affected children, as children with developmental delays are at increased risk of developing psychiatric disorders. As demonstrated with this case report, PCIT seems to be a good choice to effectively reduce disruptive behaviors in young children with partial deletion of chromosome 21q.

De novo and inherited variants in ZNF292 underlie a neurodevelopmental disorder with features of autism spectrum disorder.

PURPOSE: Intellectual disability (ID) and autism spectrum disorder (ASD) are genetically heterogeneous neurodevelopmental disorders. We sought to delineate the clinical, molecular, and neuroimaging spectrum of a novel neurodevelopmental disorder caused by variants in the zinc finger protein 292 gene (ZNF292). METHODS: We ascertained a cohort of 28 families with ID due to putatively pathogenic ZNF292 variants that were identified via targeted and exome sequencing. Available data were analyzed to characterize the canonical phenotype and examine genotype-phenotype relationships. RESULTS: Probands presented with ID as well as a spectrum of neurodevelopmental features including ASD, among others. All ZNF292 variants were de novo, except in one family with dominant inheritance. ZNF292 encodes a highly conserved zinc finger protein that acts as a transcription factor and is highly expressed in the developing human brain supporting its critical role in neurodevelopment. CONCLUSION: De novo and dominantly inherited variants in ZNF292 are associated with a range of neurodevelopmental features including ID and ASD. The clinical spectrum is broad, and most individuals present with mild to moderate ID with or without other syndromic features. Our results suggest that variants in ZNF292 are likely a recurrent cause of a neurodevelopmental disorder manifesting as ID with or without ASD.

Prenatal diagnosis of HNF1B-associated renal cysts: Is there a need to differentiate intragenic variants from 17q12 microdeletion syndrome?

OBJECTIVE: 17q12 microdeletions containing HNF1B and intragenic variants within this gene are associated with variable developmental, endocrine, and renal anomalies, often already noted prenatally as hyperechogenic/cystic kidneys. Here, we describe prenatal and postnatal phenotypes of seven individuals with HNF1B aberrations and compare their clinical and genetic data to those of previous studies. METHODS: Prenatal sequencing and postnatal chromosomal microarray analysis were performed in seven individuals with renal and/or neurodevelopmental phenotypes. We evaluated HNF1B-related clinical features from 82 studies and reclassified 192 reported intragenic HNF1B variants. RESULTS: In a prenatal case, we identified a novel in-frame deletion p.(Gly239del) within the HNF1B DNA-binding domain, a mutational hot spot as demonstrated by spatial clustering analysis and high computational prediction scores. The six postnatally diagnosed individuals harbored 17q12 microdeletions. Literature screening revealed variable reporting of HNF1B-associated clinical traits. Overall, both mutation groups showed a high phenotypic heterogeneity. The reclassification of all previously reported intragenic HNF1B variants provided an up-to-date overview of the mutational spectrum. CONCLUSIONS: We highlight the value of prenatal HNF1B screening in renal developmental diseases. Standardized clinical reporting and systematic classification of HNF1B variants are necessary for a more accurate risk quantification of prenatal and postnatal clinical features, improving genetic counseling and prenatal decision making.

Cost effectiveness of bilateral risk-reducing mastectomy and salpingo-oophorectomy.

BACKGROUND: Growing demand for risk-reducing surgery in individuals with inherited susceptibility to cancer leads to the question whether these procedures are cost effective for the executing hospitals. This study compared the clinical costs for bilateral risk-reducing mastectomy (BRRM) with and without different types of reconstruction, risk-reducing salpingo-oophorectomy (RRSO), and their combinations with corresponding reimbursements in the statutory health-care system in Germany. PATIENTS AND METHODS: Real total costs of care for BRRM with and without reconstruction, RRSO, and their combinations were calculated as the sum of all personnel and technical costs. These costs calculated in a German University hospital were compared with the sum of all reimbursements in the German DRG-based health-care system. RESULTS: While sole RRSO, BRRM without reconstruction, and BRRM with secondary DIEP (deep inferior epigastric perforator)-reconstruction still result in a small benefit, we even found shortfalls for the hospital with all other prophylactic operations under consideration. The calculated deficits were especially high for BRRM with implant-based breast reconstruction and for combined operations when the risk reduction is achieved with a minimum of separate operations. CONCLUSIONS: Risk-reducing surgery in BRCA-mutation carriers is frequently not cost-covering for the executing hospitals in the German health-care system. Thus, appropriate concepts are required to ensure a nationwide care.

Virulence Factors Produced by Staphylococcus aureus Biofilms Have a Moonlighting Function Contributing to Biofilm Integrity.

Staphylococcus aureus is the causative agent of various biofilm-associated infections in humans causing major healthcare problems worldwide. This type of infection is inherently difficult to treat because of a reduced metabolic activity of biofilm-embedded cells and the protective nature of a surrounding extracellular matrix (ECM). However, little is known about S. aureus biofilm physiology and the proteinaceous composition of the ECM. Thus, we cultivated S. aureus biofilms in a flow system and comprehensively profiled intracellular and extracellular (ECM and flow-through (FT)) biofilm proteomes, as well as the extracellular metabolome compared with planktonic cultures. Our analyses revealed the expression of many pathogenicity factors within S. aureus biofilms as indicated by a high abundance of capsule biosynthesis proteins along with various secreted virulence factors, including hemolysins, leukotoxins, and lipases as a part of the ECM. The activity of ECM virulence factors was confirmed in a hemolysis assay and a Galleria mellonella pathogenicity model. In addition, we uncovered a so far unacknowledged moonlighting function of secreted virulence factors and ribosomal proteins trapped in the ECM: namely their contribution to biofilm integrity. Mechanistically, it was revealed that this stabilizing effect is mediated by the strong positive charge of alkaline virulence factors and ribosomal proteins in an acidic ECM environment, which is caused by the release of fermentation products like formate, lactate, and acetate because of oxygen limitation in biofilms. The strong positive charge of these proteins most likely mediates electrostatic interactions with anionic cell surface components, eDNA, and anionic metabolites. In consequence, this leads to strong cell aggregation and biofilm stabilization. Collectively, our study identified a new molecular mechanism during S. aureus biofilm formation and thus significantly widens the understanding of biofilm-associated S. aureus infections - an essential prerequisite for the development of novel antimicrobial therapies.

TRIM28 haploinsufficiency predisposes to Wilms tumor.

Two percent of patients with Wilms tumors have a positive family history. In many of these cases the genetic cause remains unresolved. By applying germline exome sequencing in two families with two affected individuals with Wilms tumors, we identified truncating mutations in TRIM28. Subsequent mutational screening of germline and tumor DNA of 269 children affected by Wilms tumor was performed, and revealed seven additional individuals with germline truncating mutations, and one individual with a somatic truncating mutation in TRIM28. TRIM28 encodes a complex scaffold protein involved in many different processes, including gene silencing, DNA repair and maintenance of genomic integrity. Expression studies on mRNA and protein level showed reduction of TRIM28, confirming a loss-of-function effect of the mutations identified. The tumors showed an epithelial-type histology that stained negative for TRIM28 by immunohistochemistry. The tumors were bilateral in six patients, and 10/11 tumors are accompanied by perilobar nephrogenic rests. Exome sequencing on eight tumor DNA samples from six individuals showed loss-of-heterozygosity (LOH) of the TRIM28-locus by mitotic recombination in seven tumors, suggesting that TRIM28 functions as a tumor suppressor gene in Wilms tumor development. Additionally, the tumors showed very few mutations in known Wilms tumor driver genes, suggesting that loss of TRIM28 is the main driver of tumorigenesis. In conclusion, we identified heterozygous germline truncating mutations in TRIM28 in 11 children with mainly epithelial-type Wilms tumors, which become homozygous in tumor tissue. These data establish TRIM28 as a novel Wilms tumor predisposition gene, acting as a tumor suppressor gene by LOH.

Addition of triple negativity of breast cancer as an indicator for germline mutations in predisposing genes increases sensitivity of clinical selection criteria.

BACKGROUND: Breast cancer is the most common cancer in women. 12-15% of all tumors are triple-negative breast cancers (TNBC). So far, TNBC has been mainly associated with mutations in BRCA1. The presence of other predisposing genes seems likely since DNA damage repair is a complex process that involves several genes. Therefore we investigated if mutations in other genes are involved in cancer development and whether TNBC is an additional indicator of mutational status besides family history and age of onset. METHODS: We performed a germline panel-based screening of 10 high and low-moderate penetrance breast cancer susceptibility genes (BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, PALB2, RAD51C, RAD51D and TP53) in 229 consecutive individuals affected with TNBC unselected for age, family history or bilateral disease. Within this cohort we compared the number of mutation carriers fulfilling clinical selection criteria with the total number of carriers identified. RESULTS: Age at diagnosis ranged from 23 to 80 years with an average age of 50.2 years. In 57 women (24.9%) we detected a pathogenic mutation, with a higher frequency (29.7%) in the group manifesting cancer before 60 years. Deleterious BRCA1 mutations occurred in 14.8% of TNBC patients. These were predominantly recurrent frameshift mutations (24/34, 70.6%). Deleterious BRCA2 mutations occurred in 5.7% of patients, all but one (c.1813dupA) being unique. While no mutations were found in CDH1 and TP53, 10 mutations were detected in one of the six other predisposition genes. Remarkably, neither of the ATM, RAD51D, CHEK2 and PALB2 mutation carriers had a family history. Furthermore, patients with non-BRCA1/2 mutations were not significantly younger than mutation negative women (p = 0.3341). Most importantly, among the 57 mutation carriers, ten (17.5%) would be missed using current clinical testing criteria including five (8%) with BRCA1/2 mutations. CONCLUSIONS: In summary, our data confirm and expand previous studies of a high frequency of germline mutations in genes associated with ineffective repair of DNA damage in women with TNBCs. Neither age of onset, contralateral disease nor family history were able to discern all mutation positive individuals. Therefore, TNBC should be considered as an additional criterion for panel based genetic testing.

Risk, Prediction and Prevention of Hereditary Breast Cancer - Large-Scale Genomic Studies in Times of Big and Smart Data.

Over the last two decades genetic testing for mutations in BRCA1 and BRCA2 has become standard of care for women and men who are at familial risk for breast or ovarian cancer. Currently, genetic testing more often also includes so-called panel genes, which are assumed to be moderate-risk genes for breast cancer. Recently, new large-scale studies provided more information about the risk estimation of those genes. The utilization of information on panel genes with regard to their association with the individual breast cancer risk might become part of future clinical practice. Furthermore, large efforts have been made to understand the influence of common genetic variants with a low impact on breast cancer risk. For this purpose, almost 450 000 individuals have been genotyped for almost 500 000 genetic variants in the OncoArray project. Based on first results it can be assumed that - together with previously identified common variants - more than 170 breast cancer risk single nucleotide polymorphisms can explain up to 18% of familial breast cancer risk. The knowledge about genetic and non-genetic risk factors and its implementation in clinical practice could especially be of use for individualized prevention. This includes an individualized risk prediction as well as the individualized selection of screening methods regarding imaging and possible lifestyle interventions. The aim of this review is to summarize the most recent developments in this area and to provide an overview on breast cancer risk genes, risk prediction models and their utilization for the individual patient.

BRCA mutations and their influence on pathological complete response and prognosis in a clinical cohort of neoadjuvantly treated breast cancer patients.

PURPOSE: BRCA1/2 mutations influence the molecular characteristics and the effects of systemic treatment of breast cancer. This study investigates the impact of germline BRCA1/2 mutations on pathological complete response and prognosis in patients receiving neoadjuvant systemic chemotherapy. METHODS: Breast cancer patients were tested for a BRCA1/2 mutation in clinical routine work and were treated with anthracycline-based or platinum-based neoadjuvant chemotherapy between 1997 and 2015. These patients were identified in the tumor registry of the Breast Center of the University of Erlangen (Germany). Logistic regression and Cox regression analyses were performed to investigate the associations between BRCA1/2 mutation status, pathological complete response, disease-free survival, and overall survival. RESULTS: Among 355 patients, 59 had a mutation in BRCA1 or in BRCA2 (16.6%), 43 in BRCA1 (12.1%), and 16 in BRCA2 (4.5%). Pathological complete response defined as "ypT0; ypN0" was observed in 54.3% of BRCA1/2 mutation carriers, but only in 22.6% of non-carriers. The adjusted odds ratio was 2.48 (95% CI 1.26-4.91) for BRCA1/2 carriers versus non-carriers. Patients who achieved a pathological complete response had better disease-free survival and overall survival rates compared with those who did not achieve a pathological complete response, regardless of BRCA1/2 mutation status. CONCLUSIONS: BRCA1/2 mutation status leads to better responses to neoadjuvant chemotherapy in breast cancer. Pathological complete response is the main predictor of disease-free survival and overall survival, independently of BRCA1/2 mutation status.

Proteomic response of Streptococcus pneumoniae to iron limitation.

Iron is an essential trace element and involved in various key metabolic pathways in bacterial lifestyle. Within the human host, iron is extremely limited. Hence, the ability of bacteria to acquire iron from the environment is critical for a successful infection. Streptococcus pneumoniae (the pneumococcus) is a human pathobiont colonizing symptomless the human respiratory tract, but can also cause various local and invasive infections. To survive and proliferate pneumococci have therefore to adapt their metabolism and virulence factor repertoire to different host compartments. In this study, the response of S. pneumoniae to iron limitation as infection-relevant condition was investigated on the proteome level. The iron limitation was induced by application of the iron chelator 2,2'-bipyridine (BIP) in two different media mimicking different physiological traits. Under these conditions, the influence of the initial iron concentration on pneumococcal protein expression in response to limited iron availability was analyzed. Interestingly, one major difference between these two iron limitation experiments is the regulation of proteins involved in pneumococcal pathogenesis. In iron-poor medium several proteins of this group were downregulated whereas these proteins are upregulated in iron-rich medium. However, iron limitation in both environments led to a strong upregulation of the iron uptake protein PiuA and the significant downregulation of the non-heme iron-containing ferritin Dpr. Based on the results, it is shown that the pneumococcal proteome response to iron limitation is strongly dependent on the initial iron concentration in the medium or the environment.

Exome Pool-Seq in neurodevelopmental disorders.

High throughput sequencing has greatly advanced disease gene identification, especially in heterogeneous entities. Despite falling costs this is still an expensive and laborious technique, particularly when studying large cohorts. To address this problem we applied Exome Pool-Seq as an economic and fast screening technology in neurodevelopmental disorders (NDDs). Sequencing of 96 individuals can be performed in eight pools of 12 samples on less than one Illumina sequencer lane. In a pilot study with 96 cases we identified 27 variants, likely or possibly affecting function. Twenty five of these were identified in 923 established NDD genes (based on SysID database, status November 2016) (ACTB, AHDC1, ANKRD11, ATP6V1B2, ATRX, CASK, CHD8, GNAS, IFIH1, KCNQ2, KMT2A, KRAS, MAOA, MED12, MED13L, RIT1, SETD5, SIN3A, TCF4, TRAPPC11, TUBA1A, WAC, ZBTB18, ZMYND11), two in 543 (SysID) candidate genes (ZNF292, BPTF), and additionally a de novo loss-of-function variant in LRRC7, not previously implicated in NDDs. Most of them were confirmed to be de novo, but we also identified X-linked or autosomal-dominantly or autosomal-recessively inherited variants. With a detection rate of 28%, Exome Pool-Seq achieves comparable results to individual exome analyses but reduces costs by >85%. Compared with other large scale approaches using Molecular Inversion Probes (MIP) or gene panels, it allows flexible re-analysis of data. Exome Pool-Seq is thus well suited for large-scale, cost-efficient and flexible screening in characterized but heterogeneous entities like NDDs.

Haploinsufficiency of the Chromatin Remodeler BPTF Causes Syndromic Developmental and Speech Delay, Postnatal Microcephaly, and Dysmorphic Features.

Bromodomain PHD finger transcription factor (BPTF) is the largest subunit of nucleosome remodeling factor (NURF), a member of the ISWI chromatin-remodeling complex. However, the clinical consequences of disruption of this complex remain largely uncharacterized. BPTF is required for anterior-posterior axis formation of the mouse embryo and was shown to promote posterior neuroectodermal fate by enhancing Smad2-activated wnt8 expression in zebrafish. Here, we report eight loss-of-function and two missense variants (eight de novo and two of unknown origin) in BPTF on 17q24.2. The BPTF variants were found in unrelated individuals aged between 2.1 and 13 years, who manifest variable degrees of developmental delay/intellectual disability (10/10), speech delay (10/10), postnatal microcephaly (7/9), and dysmorphic features (9/10). Using CRISPR-Cas9 genome editing of bptf in zebrafish to induce a loss of gene function, we observed a significant reduction in head size of F0 mutants compared to control larvae. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone H3 (PH3) staining to assess apoptosis and cell proliferation, respectively, showed a significant increase in cell death in F0 mutants compared to controls. Additionally, we observed a substantial increase of the ceratohyal angle of the craniofacial skeleton in bptf F0 mutants, indicating abnormal craniofacial patterning. Taken together, our data demonstrate the pathogenic role of BPTF haploinsufficiency in syndromic neurodevelopmental anomalies and extend the clinical spectrum of human disorders caused by ablation of chromatin remodeling complexes.