Genetic Diagnostics in Routine Osteological Assessment of Adult Low Bone Mass Disorders.

CONTEXT: Many different inherited and acquired conditions can result in premature bone fragility/low bone mass disorders (LBMDs). OBJECTIVE: We aimed to elucidate the impact of genetic testing on differential diagnosis of adult LBMDs and at defining clinical criteria for predicting monogenic forms. METHODS: Four clinical centers broadly recruited a cohort of 394 unrelated adult women before menopause and men younger than 55 years with a bone mineral density (BMD) Z-score < -2.0 and/or pathological fractures. After exclusion of secondary causes or unequivocal clinical/biochemical hallmarks of monogenic LBMDs, all participants were genotyped by targeted next-generation sequencing. RESULTS: In total, 20.8% of the participants carried rare disease-causing variants (DCVs) in genes known to cause osteogenesis imperfecta (COL1A1, COL1A2), hypophosphatasia (ALPL), and early-onset osteoporosis (LRP5, PLS3, and WNT1). In addition, we identified rare DCVs in ENPP1, LMNA, NOTCH2, and ZNF469. Three individuals had autosomal recessive, 75 autosomal dominant, and 4 X-linked disorders. A total of 9.7% of the participants harbored variants of unknown significance. A regression analysis revealed that the likelihood of detecting a DCV correlated with a positive family history of osteoporosis, peripheral fractures (> 2), and a high normal body mass index (BMI). In contrast, mutation frequencies did not correlate with age, prevalent vertebral fractures, BMD, or biochemical parameters. In individuals without monogenic disease-causing rare variants, common variants predisposing for low BMD (eg, in LRP5) were overrepresented. CONCLUSION: The overlapping spectra of monogenic adult LBMD can be easily disentangled by genetic testing and the proposed clinical criteria can help to maximize the diagnostic yield.

CLN8 Mutations Presenting with a Phenotypic Continuum of Neuronal Ceroid Lipofuscinosis-Literature Review and Case Report.

CLN8 is a ubiquitously expressed membrane-spanning protein that localizes primarily in the ER, with partial localization in the ER-Golgi intermediate compartment. Mutations in CLN8 cause late-infantile neuronal ceroid lipofuscinosis (LINCL). We describe a female pediatric patient with LINCL. She exhibited a typical phenotype associated with LINCL, except she did not present spontaneous myoclonus, her symptoms occurrence was slower and developed focal sensory visual seizures. In addition, whole-exome sequencing identified a novel homozygous variant in CLN8, c.531G>T, resulting in p.Trp177Cys. Ultrastructural examination featured abundant lipofuscin deposits within mucosal cells, macrophages, and monocytes. We report a novel CLN8 mutation as a cause for NCL8 in a girl with developmental delay and epilepsy, cerebellar syndrome, visual loss, and progressive cognitive and motor regression. This case, together with an analysis of the available literature, emphasizes the existence of a continuous spectrum of CLN8-associated phenotypes rather than a sharp distinction between them.

Impaired humoral and cellular immunity after SARS-CoV-2 BNT162b2 (tozinameran) prime-boost vaccination in kidney transplant recipients.

Novel mRNA-based vaccines have been proven to be powerful tools in combating the global pandemic caused by SARS-CoV-2, with BNT162b2 (trade name: Comirnaty) efficiently protecting individuals from COVID-19 across a broad age range. Still, it remains largely unknown how renal insufficiency and immunosuppressive medication affect development of vaccine-induced immunity. We therefore comprehensively analyzed humoral and cellular responses in kidney transplant recipients after the standard second vaccination dose. As opposed to all healthy vaccinees and the majority of hemodialysis patients, only 4 of 39 and 1 of 39 transplanted individuals showed IgA and IgG seroconversion at day 8 ± 1 after booster immunization, with minor changes until day 23 ± 5, respectively. Although most transplanted patients mounted spike-specific T helper cell responses, frequencies were significantly reduced compared with those in controls and dialysis patients and this was accompanied by a broad impairment in effector cytokine production, memory differentiation, and activation-related signatures. Spike-specific CD8+ T cell responses were less abundant than their CD4+ counterparts in healthy controls and hemodialysis patients and almost undetectable in transplant patients. Promotion of anti-HLA antibodies or acute rejection was not detected after vaccination. In summary, our data strongly suggest revised vaccination approaches in immunosuppressed patients, including individual immune monitoring for protection of this vulnerable group at risk of developing severe COVID-19.

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells.

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

Longitudinal therapy monitoring of ALK-positive lung cancer by combined copy number and targeted mutation profiling of cell-free DNA.

BACKGROUND: Targeted therapies (TKI) have improved the prognosis of ALK-rearranged lung cancer (ALK+ NSCLC), but clinical courses vary widely. Early identification and molecular characterisation of treatment failure have key importance for subsequent therapies. We performed copy number variation (CNV) profiling and targeted panel sequencing from cell-free DNA (cfDNA) to monitor ALK+ NSCLC. METHODS: 271 longitudinal plasma DNA samples from 73 patients with TKI-treated metastatic ALK+ NSCLC were analysed by capture-based targeted (average coverage 4,100x), and shallow whole genome sequencing (sWGS, 0.5x). Mutations were called using standard algorithms. CNVs were quantified using the trimmed median absolute deviation from copy number neutrality (t-MAD). FINDINGS: cfDNA mutations were identified in 58% of patients. They included several potentially actionable alterations, e.g. in the genes BRAF, ERBB2, and KIT. sWGS detected CNVs in 18% of samples, compared to 6% using targeted sequencing. Several of the CNVs included potentially druggable targets, such as regions harboring EGFR, ERBB2, and MET. Circulating tumour DNA (ctDNA) mutations and t-MAD scores increased during treatment, correlated with markers of higher molecular risk, such as the EML4-ALK variant 3 and/or TP53 mutations, and were associated with shorter patient survival. Importantly, t-MAD scores reflected the tumour remission status in serial samples similar to mutant ctDNA allele frequencies, and increased with disease progression in 79% (34/43) of cases, including those without detectable single nucleotide variant (SNV). INTERPRETATION: Combined copy number and targeted mutation profiling could improve monitoring of ALK+ NSCLC. Potential advantages include the identification of treatment failure, in particular for patients without detectable mutations, and broader detection of genomic changes acquired during therapy, especially in later treatment lines and in high-risk patients. FUNDING: This work was supported by the German Center for Lung Research (DZL), by the German Cancer Consortium (DKTK), by the Heidelberg Center for Personalized Oncology at the German Cancer Research Center (DKFZ-HIPO), and by Roche Sequencing Solutions (Pleasanton, CA, USA).

Copy Number Variants Contributing to Combined Pituitary Hormone Deficiency.

Combined pituitary hormone deficiency represents a disorder with complex etiology. For many patients, causes of the disease remain unexplained, despite usage of advanced genetic testing. Although major and common transcription factors were identified two decades ago, we still struggle with identification of rare inborn factors contributing to pituitary function. In this report, we follow up genomic screening of CPHD patient cohort that were previously tested for changes in a coding sequences of genes with the use of the whole exome. We aimed to find contribution of rare copy number variations (CNVs). As a result, we identified genomic imbalances in 7 regions among 12 CPHD patients. Five out of seven regions showed copy gains whereas two presented losses of genomic fragment. Three regions with detected gains encompassed known CPHD genes namely LHX4, HESX1, and OTX2. Among new CPHD loci, the most interesting seem to be the region covering SIX3 gene, that is abundantly expressed in developing brain, and together with HESX1 contributes to pituitary organogenesis as it was evidenced before in functional studies. In conclusion, with the use of broadened genomic approach we identified copy number imbalances for 12 CPHD patients. Although further functional studies are required in order to estimate its true impact on expression pattern during pituitary organogenesis and CPHD etiology.

SEMA3A and IGSF10 Are Novel Contributors to Combined Pituitary Hormone Deficiency (CPHD).

Background: The mutation frequencies of pituitary transcription factors genes in patients with combined pituitary hormone deficiencies (CPHD) vary substantially between populations. However, apart from PROP1 the mutation rate of other genes is low and for almost half of the patients with CPHD the routine sequencing of known genes is unsuccessful in the identification of genetic causes. Methods: A cohort of 66 sporadic and nine familial CPHD cases (80 patients in total) were subjected to initial testing of the genes PROP1, POU1F1, LHX3, LHX4, and HESX1 using a targeted gene panel and MLPA. In patients who tested negative, a whole exome sequencing approach was employed. Results: In nine of the familial cases and 32 of the sporadic patients mutations in the PROP1 gene were found (the common pathogenic variants included c.301_302delAG and c.150delA). Mutations were also found in genes so far not related directly to CPHD. A unique homozygous and clinically relevant variant was identified in the SEMA3A gene, which may contribute to neural development and his phenotypic spectrum including short stature and isolated hypogonadotropic hypogonadism (IHH). Another pathogenic variant p.A1672T was found in the IGSF10 gene reported to be responsible for delayed puberty and neuronal migration during embryogenesis. Several suspected novel but predicted benign variants were also identified for the CHD7, WDR11 and FGF17 genes. Conclusion: Although PROP1 defects account for a majority of CPHD patients, identification of rare, less frequent variants constitutes a big challenge. Multiple genetic factors responsible for CPHD are still awaiting discovery and therefore the usage of efficient genomic tools (i.e., whole exome sequencing) will further broaden our knowledge regarding pituitary development and function.

The phenotype-driven computational analysis yields clinical diagnosis for patients with atypical manifestations of known intellectual disability syndromes.

BACKGROUND: Due to extensive clinical and genetic heterogeneity of intellectual disability (ID) syndromes, the process of diagnosis is very challenging even for expert clinicians. Despite recent advancements in molecular diagnostics methodologies, a significant fraction of ID patients remains without a clinical diagnosis. METHODS, RESULTS, AND CONCLUSIONS: Here, in a prospective study on a cohort of 21 families (trios) with a child presenting with ID of unknown etiology, we executed phenotype-driven bioinformatic analysis method, PhenIX, utilizing targeted next-generation sequencing (NGS) data and Human Phenotype Ontology (HPO)-encoded phenotype data. This approach resulted in clinical diagnosis for eight individuals presenting with atypical manifestations of Rubinstein-Taybi syndrome 2 (MIM 613684), Spastic Paraplegia 50 (MIM 612936), Wiedemann-Steiner syndrome (MIM 605130), Cornelia de Lange syndrome 2 (MIM 300590), Cerebral creatine deficiency syndrome 1 (MIM 300352), Glass Syndrome (MIM 612313), Mental retardation, autosomal dominant 31 (MIM 616158), and Bosch-Boonstra-Schaaf optic atrophy syndrome (MIM 615722).

Stable expansion of high-grade serous ovarian cancer organoids requires a low-Wnt environment.

High-grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the mutational profile and phenotype of the parental tumor. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for the generation of HGSOC organoids, while healthy fallopian tube organoids depend on BMP suppression by Noggin. Fallopian tube organoids modified by stable shRNA knockdown of p53, PTEN, and retinoblastoma protein (RB) also require a low-Wnt environment for long-term growth, while fallopian tube organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotypes of normal vs. loss-of-function organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation.

Serial liquid biopsies for detection of treatment failure and profiling of resistance mechanisms in KLC1-ALK-rearranged lung cancer.

Genetic rearrangements involving the anaplastic lymphoma kinase (ALK) gene confer sensitivity to ALK tyrosine kinase inhibitors (TKIs) and superior outcome in non-small-cell lung cancer (NSCLC). However, clinical courses vary widely, and recent studies suggest that molecular profiling of ALK+ NSCLC can provide additional predictors of therapy response that could assist further individualization of patient management. As repeated tissue biopsies often pose technical difficulties and significant procedural risk, analysis of tumor constituents circulating in the blood, including ctDNA and various proteins, is increasingly recognized as an alternative method of tumor sampling ("liquid biopsy"). Here, we report the case of a KLC1-ALK-rearranged NSCLC patient responding to crizotinib treatment and demonstrate how analysis of plasma and serum biomarkers can be used to identify the ALK fusion partner and monitor therapy over time. Results of ctDNA sequencing and copy-number alteration profiling as well as serum protein concentrations at various time points during therapy reflected the current remission status and could predict the subsequent clinical course. At the time of disease progression, we identified four distinct secondary mutations in the ALK gene in ctDNA potentially causing treatment failure, accompanied by rising levels of CEA and CYFRA 21-1. Moreover, several copy-number variations were detected at the end of the treatment, including an amplification of a region on Chromosome 12 encompassing the TP53 regulator MDM2 In summary, our findings illustrate the utility of noninvasive longitudinal molecular profiling for assessing remission status, exploring mechanisms of treatment failure, predicting subsequent clinical course, and dissecting dynamics of drug-resistant clones in ALK+ lung cancer.

Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties.

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics.

Detection of TP53 Mutations in Tissue or Liquid Rebiopsies at Progression Identifies ALK+ Lung Cancer Patients with Poor Survival.

Anaplastic lymphoma kinase (ALK) sequencing can identify resistance mechanisms and guide next-line therapy in ALK+ non-small-cell lung cancer (NSCLC), but the clinical significance of other rebiopsy findings remains unclear. We analysed all stage-IV ALK+ NSCLC patients with longitudinally assessable TP53 status treated in our institutions (n = 62). Patients with TP53 mutations at baseline (TP53mutbas, n = 23) had worse overall survival (OS) than patients with initially wild-type tumours (TP53wtbas, n = 39, 44 vs. 62 months in median, p = 0.018). Within the generally favourable TP53wtbas group, detection of TP53 mutations at progression defined a "converted" subgroup (TP53mutconv, n = 9) with inferior OS, similar to that of TP53mutbas and shorter than that of patients remaining TP53 wild-type (TP53wtprogr, 45 vs. 94 months, p = 0.043). Progression-free survival (PFS) under treatment with tyrosine kinase inhibitors (TKI) for TP53mutconv was comparable to that of TP53mutbas and also shorter than that of TP53wtprogr cases (5 and 8 vs. 13 months, p = 0.0039). Fewer TP53wtprogr than TP53mutbas or TP53mutconv cases presented with metastatic disease at diagnosis (67% vs. 91% or 100%, p < 0.05). Thus, acquisition of TP53 mutations at progression is associated with more aggressive disease, shorter TKI responses and inferior OS in ALK+ NSCLC, comparable to primary TP53 mutated cases.

Genomic landscape and clonal evolution of acute myeloid leukemia with t(8;21): an international study on 331 patients.

Acute myeloid leukemia with t(8;21)(q22;q22) is characterized by considerable clinical and biological heterogeneity leading to relapse in up to 40% of patients. We sequenced coding regions or hotspot areas of 66 recurrently mutated genes in a cohort of 331 t(8;21) patients. At least 1 mutation, in addition to t(8;21), was identified in 95%, with a mean of 2.2 driver mutations per patient. Recurrent mutations occurred in genes related to RAS/RTK signaling (63.4%), epigenetic regulators (45%), cohesin complex (13.6%), MYC signaling (10.3%), and the spliceosome (7.9%). Our study identified mutations in previously unappreciated genes: GIGYF2, DHX15, and G2E3 Based on high mutant levels, pairwise precedence, and stability at relapse, epigenetic regulator mutations were likely to occur before signaling mutations. In 34% of RAS/RTKmutated patients, we identified multiple mutations in the same pathway. Deep sequencing (∼42 000×) of 126 mutations in 62 complete remission samples from 56 patients identified 16 persisting mutations in 12 patients, of whom 5 lacked RUNX1-RUNX1T1 in quantitative polymerase chain reaction analysis. KIT high mutations defined by a mutant level ≥25% were associated with inferior relapse-free survival (hazard ratio, 1.96; 95% confidence interval, 1.22-3.15; P = .005). Together with age and white blood cell counts, JAK2, FLT3-internal tandem duplicationhigh, and KIT high mutations were identified as significant prognostic factors for overall survival in multivariate analysis. Whole-exome sequencing was performed on 19 paired diagnosis, remission, and relapse trios. Exome-wide analysis showed an average of 16 mutations with signs of substantial clonal evolution. Based on the resemblance of diagnosis and relapse pairs, genetically stable (n = 13) and unstable (n = 6) subgroups could be identified.

Role of Donor Clonal Hematopoiesis in Allogeneic Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clonal hematopoiesis of indeterminate potential (CHIP) occurs in the blood of approximately 20% of older persons. CHIP is linked to an increased risk of hematologic malignancies and of all-cause mortality; thus, the eligibility of stem-cell donors with CHIP is questionable. We comprehensively investigated how donor CHIP affects outcome of allogeneic hematopoietic stem-cell transplantation (HSCT). METHODS: We collected blood samples from 500 healthy, related HSCT donors (age ≥ 55 years) at the time of stem-cell donation for targeted sequencing with a 66-gene panel. The effect of donor CHIP was assessed on recipient outcomes, including graft-versus-host disease (GVHD), cumulative incidence of relapse/progression (CIR/P), and overall survival (OS). RESULTS: A total of 92 clonal mutations with a median variant allele frequency of 5.9% were identified in 80 (16.0%) of 500 donors. CHIP prevalence was higher in donors related to patients with myeloid compared with lymphoid malignancies (19.2% v 6.3%; P ≤ .001). In recipients allografted with donor CHIP, we found a high cumulative incidence of chronic GVHD (cGVHD; hazard ratio [HR], 1.73; 95% CI, 1.21 to 2.49; P = .003) and lower CIR/P (univariate: HR, 0.62; 95% CI, 0.40 to 0.97; P = .027; multivariate: HR, 0.63; 95% CI, 0.41 to 0.98; P = .042) but no effect on nonrelapse mortality. Serial quantification of 25 mutations showed engraftment of 24 of 25 clones and disproportionate expansion in half of them. Donor-cell leukemia was observed in two recipients. OS was not affected by donor CHIP status (HR, 0.88; 95% CI, 0.65 to 1.321; P = .434). CONCLUSION: Allogeneic HSCT from donors with CHIP seems safe and results in similar survival in the setting of older, related donors. Future studies in younger and unrelated donors are warranted to extend these results. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that donor CHIP might foster cGVHD development and reduce relapse/progression risk.

Hematopoietic lineage distribution and evolutionary dynamics of clonal hematopoiesis.

Clonal hematopoiesis of indeterminate potential (CHIP) occurs in an age-related manner and associates with an increased risk of hematologic cancer, atherosclerotic disease, and shorter overall survival. Little is known about the cell of origin, repartition patterns of clonal mutations within the hematopoietic differentiation tree, and its dynamics under evolutionary pressure. Using targeted sequencing, CHIP was identified in 121 out of 437 elderly individuals (27.7%). Variant allele frequencies (VAFs) of 91 mutations were studied in six peripheral blood cell fractions. VAFs were significantly higher in monocytes, granulocytes, and NK-cells compared to B- or T cells. In all cases with available bone marrow material, mutations could be identified in Lin-CD34+CD38- HSCs with subsequent expansion to myeloid primed progenitors. In 22 patients with solid cancer receiving (radio-)chemotherapy, longitudinal study of 32 mutations at 121 time points identified relative VAF changes of at least 50% in 13/32 mutations. VAFs of DNMT3A, were stable in 12/13 cases (P < .001). Cancer patients with a clonal mutation other than DNMT3A required more often red blood cell transfusions and dose reductions. Our results provide novel insights into cellular distribution of clonal mutations, their dynamics under chemotherapy, and advocate for systematic analyses for CHIP in cancer patients.

Ocular findings in Loeys-Dietz syndrome.

BACKGROUND: Loeys-Dietz syndrome (LDS), an autosomal-dominant connective tissue disorder, is characterised by systemic manifestations including arterial aneurysm and craniofacial dysmorphologies. Although ocular involvement in LDS has been reported, detailed information on those manifestations is lacking. METHODS: Retrospective chart review of patients with diagnosed LDS and comparison with age-matched control patients. RESULTS: Mean age was 37.8±14.6 years (patients with LDS) and 38.4±13.5 years (controls). Patients with LDS less frequently had iris transillumination, cataract and glaucoma compared with controls. Scleral and retinal vascular abnormalities were not found in any of the LDS eyes. Ectopia lentis was found in one patient with LDS. The eyes of patients with LDS tended to be more myopic (spherical equivalent, -2.47±2.70 dioptres (dpt) vs -1.30±2.96dpt (controls); P=0.08) and longer (24.6±1.7mm vs 24.1±1.5mm (controls); P=0.10). Central corneal thickness was significantly reduced in LDS eyes (521±48µm vs 542±37µm (controls); P=0.02). Corneal curvature (43.06±1.90dpt (LDS) versus 43.00±1.37dpt (controls); P=0.72) and interpupillary distance (65.0±6.0mm (LDS) vs 64.3±4.8mm (controls); P=0.66) did not differ significantly between both groups. Visual acuity was similar between both groups (0.03±0.09logarithm of the minimum angle of resolution (logMAR) for LDS eyes and 0.05±0.17logMAR for control eyes, P=0.47). CONCLUSIONS: Ocular features of LDS include decreased central corneal thickness and mild myopia. Ectopia lentis may be slightly more common than in controls but appears less common than in Marfan syndrome. Hypertelorism, scleral and retinal vascular abnormalities were not features of LDS.

The germline variants in DNA repair genes in pediatric medulloblastoma: a challenge for current therapeutic strategies.

BACKGROUND: The defects in DNA repair genes are potentially linked to development and response to therapy in medulloblastoma. Therefore the purpose of this study was to establish the spectrum and frequency of germline variants in selected DNA repair genes and their impact on response to chemotherapy in medulloblastoma patients. METHODS: The following genes were investigated in 102 paediatric patients: MSH2 and RAD50 using targeted gene panel sequencing and NBN variants (p.I171V and p.K219fs*19) by Sanger sequencing. In three patients with presence of rare life-threatening adverse events (AE) and no detected variants in the analyzed genes, whole exome sequencing was performed. Based on combination of molecular and immunohistochemical evaluations tumors were divided into molecular subgroups. Presence of variants was tested for potential association with the occurrence of rare life-threatening AE and other clinical features. RESULTS: We have identified altogether six new potentially pathogenic variants in MSH2 (p.A733T and p.V606I), RAD50 (p.R1093*), FANCM (p.L694*), ERCC2 (p.R695C) and EXO1 (p.V738L), in addition to two known NBN variants. Five out of twelve patients with defects in either of MSH2, RAD50 and NBN genes suffered from rare life-threatening AE, more frequently than in control group (p = 0.0005). When all detected variants were taken into account, the majority of patients (8 out of 15) suffered from life-threatening toxicity during chemotherapy. CONCLUSION: Our results, based on the largest systematic study performed in a clinical setting, provide preliminary evidence for a link between defects in DNA repair genes and treatment related toxicity in children with medulloblastoma. The data suggest that patients with DNA repair gene variants could need special vigilance during and after courses of chemotherapy.

Mutations in proteasome-related genes are associated with thyroid hemiagenesis.

PURPOSE: Human thyroid development is a complex and still unexplained process. Thyroid hemiagenesis is a congenital anomaly, where one of the thyroid lobes fails to develop. In the majority of patients with thyroid hemiagenesis, the genetic background remains unknown. The aim of the study was to search for novel genetic contributors to the etiology of thyroid hemiagenesis. METHODS: A cohort of 34 sporadic patients diagnosed with thyroid hemiagenesis and one three-generation family were subjected to comprehensive genomic examination. Initially, targeted screening of associated transcription factors, known to be linked to thyroid development, was performed. As a next step, genomic examinations were applied using high-resolution microarrays, whereas for the thyroid hemiagenesis family, additionally the whole exome sequencing was performed. RESULTS: Screening of transcription factors revealed no causative mutations in the studied cohort. Genomic examinations revealed the presence of four recurrent defects (three deletions and one duplication) affecting highly conservative proteasome genes PSMA1, PSMA3, and PSMD3. In a thyroid hemiagenesis family a splice site mutation in a proteasome gene PSMD2 (c.612T > C cDNA.1170T > C, g.3271T > C) was found in both affected mother and daughter. CONCLUSIONS: Our results shed a new light on etiology of thyroid hemiagenesis, so far suspected to be linked only to mutations in the genes directly involved in the thyroid development. We demonstrated, for the first time, that genomic alterations in proteasome-associated genes co-occur in patients presenting this developmental anomaly.

L1Base 2: more retrotransposition-active LINE-1s, more mammalian genomes.

LINE-1 (L1) insertions comprise as much as 17% of the human genome sequence, and similar proportions have been recorded for other mammalian species. Given the established role of L1 retrotransposons in shaping mammalian genomes, it becomes an important task to track and annotate the sources of this activity: full length elements, able to encode the cis and trans acting components of the retrotransposition machinery. The L1Base database (http://l1base.charite.de) contains annotated full-length sequences of LINE-1 transposons including putatively active L1s. For the new version of L1Base, a LINE-1 annotation tool, L1Xplorer, has been used to mine potentially active L1 retrotransposons from the reference genome sequences of 17 mammals. The current release of the human genome, GRCh38, contains 146 putatively active L1 elements or full length intact L1 elements (FLIs). The newest versions of the mouse, GRCm38 and the rat, Rnor_6.0, genomes contain 2811 and 492 FLIs, respectively. Most likely reflecting the current level of completeness of the genome project, the latest reference sequence of the common chimpanzee genome, PT 2.19, only contains 19 FLIs. Of note, the current assemblies of the dog, CF 3.1 and the sheep, OA 3.1, genomes contain 264 and 598 FLIs, respectively. Further developments in the new version of L1Base include an updated website with implementation of modern web server technologies. including a more responsive design for an improved user experience, as well as the addition of data sharing capabilities for L1Xplorer annotation.