Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase.

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

De novo variants in DENND5B cause a neurodevelopmental disorder.

The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement.

Identifying digenic disease genes via machine learning in the Undiagnosed Diseases Network.

Rare diseases affect millions of people worldwide, and discovering their genetic causes is challenging. More than half of the individuals analyzed by the Undiagnosed Diseases Network (UDN) remain undiagnosed. The central hypothesis of this work is that many of these rare genetic disorders are caused by multiple variants in more than one gene. However, given the large number of variants in each individual genome, experimentally evaluating combinations of variants for potential to cause disease is currently infeasible. To address this challenge, we developed the digenic predictor (DiGePred), a random forest classifier for identifying candidate digenic disease gene pairs by features derived from biological networks, genomics, evolutionary history, and functional annotations. We trained the DiGePred classifier by using DIDA, the largest available database of known digenic-disease-causing gene pairs, and several sets of non-digenic gene pairs, including variant pairs derived from unaffected relatives of UDN individuals. DiGePred achieved high precision and recall in cross-validation and on a held-out test set (PR area under the curve > 77%), and we further demonstrate its utility by using digenic pairs from the recent literature. In contrast to other approaches, DiGePred also appropriately controls the number of false positives when applied in realistic clinical settings. Finally, to enable the rapid screening of variant gene pairs for digenic disease potential, we freely provide the predictions of DiGePred on all human gene pairs. Our work enables the discovery of genetic causes for rare non-monogenic diseases by providing a means to rapidly evaluate variant gene pairs for the potential to cause digenic disease.

Prevalence of Individuals With Multiple Diagnosed Genetic Diseases in the Undiagnosed Diseases Network.

Report the prevalence of multiple genetic diseases in the Undiagnosed Diseases Network (UDN) cohort in the post-exome-sequencing era. UDN subjects underwent genome sequencing before inclusion in the cohort. Records of all UDN subjects until January 2024 were analyzed. The number of diagnoses, proportion of molecular versus nonmolecular (i.e., not attributable to a discretely identifiable genetic change) diagnoses, and the inheritance patterns of the genetic diagnoses were determined. Of 2799 subjects, 766 (27.4%) had diagnoses. Of these 766, 95.4% had one diagnosis, 4.0% had two diagnoses, and 0.5% had three diagnoses. Of the diagnosed subjects, 93.4% had a genetic disease, and 6.5% had a nonmolecular disease. Of subjects with two diagnoses, both diagnoses were molecular in 90.3%, while 9.7% had one molecular and one nonmolecular diagnosis. All four subjects with three diagnoses had three molecular diagnoses. 4.2% of diagnosed subjects in the UDN had more than one molecular diagnosis, with four individuals having three concurrent Mendelian diagnoses. Additionally, three subjects had concurrent molecular and nonmolecular diagnoses. Given that numerous UDN subjects had a negative genome sequence prior to UDN enrollment, multiple molecular diagnoses may contribute to diagnostic uncertainty even with genome sequencing, as may concurrent nonmolecular disease.

Data from electronic healthcare records expand our understanding of X-linked genetic diseases.

Disease specific cohort studies have reported details on X linked (XL) disorders affecting females. We investigated the spectrum and penetrance of XL disorders seen in electronic health records (EHR). We generated a cohort of individuals diagnosed with XL disorders at Vanderbilt University Medical Center over 20 years. Our cohort included 477 males and 203 females diagnosed with 108 different XL genetic disorders. We found large differences between the female/male (F/M) ratios for various XL disorders regardless of their OMIM annotated mode of inheritance. We identified four XL recessive disorders affecting women previously only described in men. Biomarkers for XL disease had unique gender-specific patterns differing between modes of inheritance. EHRs provide large cohorts of XL genetic disorders that give new insights compared to the literature. Differences in the F/M ratios and biomarkers of XL disorders observed likely result from disease specific and sex dependent penetrance. We conclude that observed gender ratios associated with specific XL disorders may be more useful than those predicted by Mendelian genetics provided by OMIM. Our findings of a gender specific penetrance and severity for XL disorders show unexpected differences from Mendelian predictions. Further work is required to validate our findings in larger combined EHR cohorts.

Probable digenic inheritance of Diamond-Blackfan anemia.

A 26-year-old female proband with a clinical diagnosis and consistent phenotype of Diamond-Blackfan anemia (DBA, OMIM 105650) without an identified genotype was referred to the Undiagnosed Diseases Network. DBA is classically associated with monoallelic variants that have an autosomal-dominant or -recessive mode of inheritance. Intriguingly, her case was solved by a detection of a digenic interaction between non-allelic RPS19 and RPL27 variants. This was confirmed with a machine learning structural model, co-segregation analysis, and RNA sequencing. This is the first report of DBA caused by a digenic effect of two non-allelic variants demonstrated by machine learning structural model. This case suggests that atypical phenotypic presentations of DBA may be caused by digenic inheritance in some individuals. We also conclude that a machine learning structural model can be useful in detecting digenic models of possible interactions between products encoded by alleles of different genes inherited from non-affected carrier parents that can result in DBA with an unrealized 25% recurrence risk.

Advancing Equity in Rare Disease Research: Insights From the Undiagnosed Disease Network.

Rare diseases affect 6%-8% of the population and present diagnostic challenges, particularly for historically marginalized ethnic and racial groups. The Undiagnosed Diseases Network (UDN) aims to enhance diagnosis rates and research participation among such minoritized groups. A retrospective review was conducted from 2015 to 2023, analyzing 2235 UDN participants to evaluate its progress toward this objective. Data on demographics, disease phenotypes, diagnostic outcomes, and socioeconomic factors were collected and statistical analyses assessed differences among ethnic and racial groups. This demonstrated that Hispanic and Black non-Hispanic groups were underrepresented, while White non-Hispanic participants were overrepresented in the UDN compared to the US population. Individuals whose primary language was not English were also significantly underrepresented. Diagnosis rates varied, with the highest rates among Asian non-Hispanic (39.5%) and Hispanic (35.3%) groups and the lowest rate in the White non-Hispanic group (26.8%) (p < 0.001). Binomial logistic regression found, however, that only participant age and disease phenotype predicted the likelihood of receiving a diagnosis (p < 0.001). Persistent ethnic and racial disparities in UDN participation appear to be associated with major differences in application rates. Under-enrollment of historically marginalized ethnic and racial groups may be due to economic hardships and language barriers. No differences in the diagnostic yield among ethnic and racial groups were observed after controlling for other factors. This work highlights the value of comprehensive genetic evaluations for addressing healthcare disparities and suggests priorities for advancing inclusion in rare disease research.

Undiagnosed Disease Network collaborative approach in diagnosing rare disease in a patient with a mosaic CACNA1D variant.

The Undiagnosed Disease Network (UDN) is comprised of clinical and research experts collaborating to diagnose rare disease. The UDN is funded by the National Institutes of Health and includes 12 different clinical sites (About Us, 2022). Here we highlight the success of collaborative efforts within the UDN Clinical Site at Vanderbilt University Medical Center (VUMC) in utilizing a cohort of experts in bioinformatics, structural biology, and genetics specialists in diagnosing rare disease. Our UDN team identified a de novo mosaic CACNA1D variant c.2299T>C in a 5-year-old female with a history of global developmental delay, dystonia, dyskinesis, and seizures. Using a collaborative multidisciplinary approach, our VUMC UDN team diagnosed the participant with Primary Aldosteronism, Seizures, and Neurologic abnormalities (PASNA) OMIM: 615474 due to a rare mosaic CACNA1D variant (O'Neill, 2013). Interestingly, this patient was mosaic, a phenotypic trait previously unreported in PASNA cases. This report highlights the importance of a multidisciplinary approach in diagnosing rare disease.

The Genetic Landscape of Familial Pulmonary Fibrosis.

Rationale and Objectives: Up to 20% of idiopathic interstitial lung disease is familial, referred to as familial pulmonary fibrosis (FPF). An integrated analysis of FPF genetic risk was performed by comprehensively evaluating for genetic rare variants (RVs) in a large cohort of FPF kindreds. Methods: Whole-exome sequencing and/or candidate gene sequencing from affected individuals in 569 FPF kindreds was performed, followed by cosegregation analysis in large kindreds, gene burden analysis, gene-based risk scoring, cell-type enrichment analysis, and coexpression network construction. Measurements and Main Results: It was found that 14.9-23.4% of genetic risk in kindreds could be explained by RVs in genes previously linked to FPF, predominantly telomere-related genes. New candidate genes were identified in a small number of families-including SYDE1, SERPINB8, GPR87, and NETO1-and tools were developed for evaluation and prioritization of RV-containing genes across kindreds. Several pathways were enriched for RV-containing genes in FPF, including focal adhesion and mitochondrial complex I assembly. By combining single-cell transcriptomics with prioritized candidate genes, expression of RV-containing genes was discovered to be enriched in smooth muscle cells, type II alveolar epithelial cells, and endothelial cells. Conclusions: In the most comprehensive FPF genetic study to date, the prevalence of RVs in known FPF-related genes was defined, and new candidate genes and pathways relevant to FPF were identified. However, new RV-containing genes shared across multiple kindreds were not identified, thereby suggesting that heterogeneous genetic variants involving a variety of genes and pathways mediate genetic risk in most FPF kindreds.

Changing Nepali children's attitudes toward conservation through a participatory education program.

People's attitude toward wildlife is known to affect the success of conservation programs. Public education can increase support for conservation measures by providing the knowledge necessary to make informed decisions, improve pro-environmental behaviour and potentially enlist participation. To that end, we developed a participatory conservation education program in Nepal with a target audience of secondary school students. Our lessons emphasized ecological balance by highlighting the roles of individual species, including the rhesus macaque (Macaca mulatta). This species presents a challenge for educating the public about maintaining balance in nature, because although an important seed disperser and species of religious significance, macaques are frequently involved in human-wildlife conflict and considered a pest species. We evaluated the impact of our program by surveying students from six socioeconomically similar schools, four that participated in our program and two that had not. Our questionnaire was comprised of knowledge questions about local wildlife and conservation issues, an open-ended listing exercise to determine species preferences, and statements that elicited students' opinions regarding empathy toward animals. Knowledge about and attitude toward wildlife, including M. mulatta, were greater in students that participated in the program. More importantly, these students were more likely to understand the role individual species play in a balanced ecosystem, how human behaviour can negatively impact wildlife, and how they could participate in conservation efforts. Gender was not a factor in any response. We discuss the merits of using a participatory learning platform in a standalone education program that has long-term purpose, funding, and buy-in.

Rare structural variants, aneuploidies, and mosaicism in individuals with Mullerian aplasia detected by optical genome mapping.

The molecular basis of Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome remains largely unknown. Pathogenic variants in WNT4 and HNF1B have been confirmed in a small percent of individuals. A variety of copy number variants have been reported, but causal gene(s) remain to be identified. We hypothesized that rare structural variants (SVs) would be present in some individuals with MRKH, which could explain the genetic basis of the syndrome. Large molecular weight DNA was extracted from lymphoblastoid cells from 87 individuals with MRKH and available parents. Optical genome mapping (OGM) was performed to identify SVs, which were confirmed by another method (quantitative PCR, chromosomal microarray, karyotype, or fluorescent in situ hybridization) when possible. Thirty-four SVs that overlapped coding regions of genes with potential involvement in MRKH were identified, 14 of which were confirmed by a second method. These 14 SVs were present in 17/87 (19.5%) of probands with MRKH and included seven deletions, three duplications, one new translocation in 5/50 cells-t(7;14)(q32;q32), confirmation of a previously identified translocation-t(3;16)(p22.3;p13.3), and two aneuploidies. Of interest, three cases of mosaicism (3.4% of probands) were identified-25% mosaicism for trisomy 12, 45,X(75%)/46,XX (25%), and 10% mosaicism for a 7;14 translocation. Our study constitutes the first systematic investigation of SVs by OGM in individuals with MRKH. We propose that OGM is a promising method that enables a comprehensive investigation of a variety of SVs in a single assay including cryptic translocations and mosaic aneuploidies. These observations suggest that mosaicism could play a role in the genesis of MRKH.

A medical odyssey of a 72-year-old man with Charcot-Marie-Tooth disease type 2 newly diagnosed with biallelic variants in SORD gene causing sorbitol dehydrogenase deficiency.

A 72-year-old man was referred to the Undiagnosed Diseases Network (UDN) because of gradual progressive weakness in both lower extremities for the past 45 years. He was initially diagnosed as having Charcot-Marie-Tooth disease type 2 (CMT2) without a defined molecular genetic cause. Exome sequencing (ES) failed to detect deleterious neuromuscular variants. Very recently, biallelic variants in sorbitol dehydrogenase (SORD) were discovered to be a novel cause of inherited neuropathies including CMT2 or distal hereditary motor neuropathy (dHMN) referred to as Sorbitol Dehydrogenase Deficiency with Peripheral Neuropathy (SORDD, OMIM 618912). The most common variant identified was c.757delG; p.A253Qfs*27. Through the Vanderbilt UDN clinical site, this patient was formally diagnosed with SORDD after the identification of homozygosity for the above SORD frameshift through UDN Genome Sequencing (GS). His medical odyssey was solved by GS and detection of extremely high levels of sorbitol. The diagnosis provided him the opportunity to receive potential treatment with an investigational drug in a clinical trial for SORDD. We suggest that similar studies be considered in other individuals thought to possibly have CMT2 or dHMN.

The contribution of mosaicism to genetic diseases and de novo pathogenic variants.

The contribution of mosaicism to diagnosed genetic disease and presumed de novo variants (DNV) is under investigated. We determined the contribution of mosaic genetic disease (MGD) and diagnosed parental mosaicism (PM) in parents of offspring with reported DNV (in the same variant) in the (1) Undiagnosed Diseases Network (UDN) (N = 1946) and (2) in 12,472 individuals electronic health records (EHR) who underwent genetic testing at an academic medical center. In the UDN, we found 4.51% of diagnosed probands had MGD, and 2.86% of parents of those with DNV exhibited PM. In the EHR, we found 6.03% and 2.99% and (of diagnosed probands) had MGD detected on chromosomal microarray and exome/genome sequencing, respectively. We found 2.34% (of those with a presumed pathogenic DNV) had a parent with PM for the variant. We detected mosaicism (regardless of pathogenicity) in 4.49% of genetic tests performed. We found a broad phenotypic spectrum of MGD with previously unknown phenotypic phenomena. MGD is highly heterogeneous and provides a significant contribution to genetic diseases. Further work is required to improve the diagnosis of MGD and investigate how PM contributes to DNV risk.

A recurrent single-exon deletion in TBCK might be under-recognized in patients with infantile hypotonia and psychomotor delay.

Advanced bioinformatics algorithms allow detection of multiple-exon copy-number variations (CNVs) from exome sequencing (ES) data, while detection of single-exon CNVs remains challenging. A retrospective review of Baylor Genetics' clinical ES patient cohort identified four individuals with homozygous single-exon deletions of TBCK (exon 23, NM_001163435.2), a gene associated with an autosomal recessive neurodevelopmental phenotype. To evaluate the prevalence of this deletion and its contribution to disease, we retrospectively analyzed single nucleotide polymorphism (SNP) array data for 8194 individuals undergoing ES, followed by PCR confirmation and RT-PCR on individuals carrying homozygous or heterozygous exon 23 TBCK deletions. A fifth individual was diagnosed with the TBCK-related disorder due to a heterozygous exon 23 deletion in trans with a c.1860+1G>A (NM_001163435.2) pathogenic variant, and three additional heterozygous carriers were identified. Affected individuals and carriers were from diverse ethnicities including European Caucasian, South Asian, Middle Eastern, Hispanic American and African American, with only one family reporting consanguinity. RT-PCR revealed two out-of-frame transcripts related to the exon 23 deletion. Our results highlight the importance of identifying single-exon deletions in clinical ES, especially for genes carrying recurrent deletions. For patients with early-onset hypotonia and psychomotor delay, this single-exon TBCK deletion might be under-recognized due to technical limitations of ES.

Efficacy of virtual and asynchronous teaching of computer-assisted diagnosis of genetic diseases seen in clinics.

We studied if clinicians could gain sufficient working knowledge of a computer-assisted diagnostic decision support system (DDSS) (SimulConsult), to make differential diagnoses (DDx) of genetic disorders. We hypothesized that virtual training could be convenient, asynchronous, and effective in teaching clinicians how to use a DDSS. We determined the efficacy of virtual, asynchronous teaching for clinicians to gain working knowledge to make computer-assisted DDx. Our study consisted of three surveys (Baseline, Training, and After Use) and a series of case problems sent to clinicians at Vanderbilt University Medical Center. All participants were able to generate computer-assisted DDx that achieved passing scores of the case problems. Between 75% and 92% agreed/completely agreed the DDSS was useful to their work and for clinical decision support and was easy to use. Participants' use of the DDSS resulted in statistically significant time savings in key tasks and in total time spent on clinical tasks. Our results indicate that virtual, asynchronous teaching can be an effective format to gain a working knowledge of a DDSS, and its clinical use could result in significant time savings across multiple tasks as well as facilitate synergistic interaction between clinicians and lab specialists. This approach is especially pertinent and offers value amid the COVID-19 pandemic.

Genetics of agenesis/hypoplasia of the uterus and vagina: narrowing down the number of candidate genes for Mayer-Rokitansky-Küster-Hauser Syndrome.

PURPOSE: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome consists of congenital absence of the uterus and vagina and is often associated with renal, skeletal, cardiac, and auditory defects. The genetic basis is largely unknown except for rare variants in several genes. Many candidate genes have been suggested by mouse models and human studies. The purpose of this study was to narrow down the number of candidate genes. METHODS: Whole exome sequencing was performed on 111 unrelated individuals with MRKH; variant analysis focused on 72 genes suggested by mouse models, human studies of physiological candidates, or located near translocation breakpoints in t(3;16). Candidate variants (CV) predicted to be deleterious were confirmed by Sanger sequencing. RESULTS: Sanger sequencing verified 54 heterozygous CV from genes identified through mouse (13 CV in 6 genes), human (22 CV in seven genes), and translocation breakpoint (19 CV in 11 genes) studies. Twelve patients had ≥ 2 CVs, including four patients with two variants in the same gene. One likely digenic combination of LAMC1 and MMP14 was identified. CONCLUSION: We narrowed 72 candidate genes to 10 genes that appear more likely implicated. These candidate genes will require further investigation to elucidate their role in the development of MRKH.

A Recurrent De Novo Heterozygous COG4 Substitution Leads to Saul-Wilson Syndrome, Disrupted Vesicular Trafficking, and Altered Proteoglycan Glycosylation.

The conserved oligomeric Golgi (COG) complex is involved in intracellular vesicular transport, and is composed of eight subunits distributed in two lobes, lobe A (COG1-4) and lobe B (COG5-8). We describe fourteen individuals with Saul-Wilson syndrome, a rare form of primordial dwarfism with characteristic facial and radiographic features. All affected subjects harbored heterozygous de novo variants in COG4, giving rise to the same recurrent amino acid substitution (p.Gly516Arg). Affected individuals' fibroblasts, whose COG4 mRNA and protein were not decreased, exhibited delayed anterograde vesicular trafficking from the ER to the Golgi and accelerated retrograde vesicular recycling from the Golgi to the ER. This altered steady-state equilibrium led to a decrease in Golgi volume, as well as morphologic abnormalities with collapse of the Golgi stacks. Despite these abnormalities of the Golgi apparatus, protein glycosylation in sera and fibroblasts from affected subjects was not notably altered, but decorin, a proteoglycan secreted into the extracellular matrix, showed altered Golgi-dependent glycosylation. In summary, we define a specific heterozygous COG4 substitution as the molecular basis of Saul-Wilson syndrome, a rare skeletal dysplasia distinct from biallelic COG4-CDG.

Effect of Genetic Diagnosis on Patients with Previously Undiagnosed Disease.

BACKGROUND: Many patients remain without a diagnosis despite extensive medical evaluation. The Undiagnosed Diseases Network (UDN) was established to apply a multidisciplinary model in the evaluation of the most challenging cases and to identify the biologic characteristics of newly discovered diseases. The UDN, which is funded by the National Institutes of Health, was formed in 2014 as a network of seven clinical sites, two sequencing cores, and a coordinating center. Later, a central biorepository, a metabolomics core, and a model organisms screening center were added. METHODS: We evaluated patients who were referred to the UDN over a period of 20 months. The patients were required to have an undiagnosed condition despite thorough evaluation by a health care provider. We determined the rate of diagnosis among patients who subsequently had a complete evaluation, and we observed the effect of diagnosis on medical care. RESULTS: A total of 1519 patients (53% female) were referred to the UDN, of whom 601 (40%) were accepted for evaluation. Of the accepted patients, 192 (32%) had previously undergone exome sequencing. Symptoms were neurologic in 40% of the applicants, musculoskeletal in 10%, immunologic in 7%, gastrointestinal in 7%, and rheumatologic in 6%. Of the 382 patients who had a complete evaluation, 132 received a diagnosis, yielding a rate of diagnosis of 35%. A total of 15 diagnoses (11%) were made by clinical review alone, and 98 (74%) were made by exome or genome sequencing. Of the diagnoses, 21% led to recommendations regarding changes in therapy, 37% led to changes in diagnostic testing, and 36% led to variant-specific genetic counseling. We defined 31 new syndromes. CONCLUSIONS: The UDN established a diagnosis in 132 of the 382 patients who had a complete evaluation, yielding a rate of diagnosis of 35%. (Funded by the National Institutes of Health Common Fund.).

Clinical diagnosis of neurofibromatosis type I in multiple family members due to cosegregation of a unique balanced translocation with disruption of the NF1 locus: Testing considerations for accurate diagnosis.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that causes a predisposition to develop tumors along the peripheral nervous system. The NF1 gene, located at 17q11.2, has the highest mutation rate among known human genes and about half of NF1 patients have de novo pathogenic variants. We present a case of clinical NF1 diagnoses in multiple family members with phenotypes ranging from mild to severe. Chromosome analysis of the 3-year-old female proband with NF1 resulted in an abnormal karyotype that was inherited from her mother: 46,XX,t(4;17)(q21.3;q11.2) mat. However, no NF1 genetic variants were identified by either NGS analysis of NF1 DNA coding regions, deletion-duplication studies, or by cytogenomic microarray copy number analysis. Follow-up chromosome studies of the proband's two male siblings demonstrated cosegregation of the same balanced translocation and a clinical diagnosis of NF1. Based on the cosegregation of the translocation with the NF1 clinical presentation in this family, we hypothesized that the NF1 gene may have been disrupted by this unique rearrangement. Subsequent fluorescence in situ hybridization (FISH) analysis of the metaphase cells of an affected sibling revealed a disruption of the NF1 gene confirming the underlying basis of the clinical NF1 presentation in this family. The utilization of traditional cytogenetic as well as evolving molecular methods was not only pivotal in the diagnosis of NF1 and management for this family, but is also pertinent to other patients with a family history of NF1.

Histopathologic analysis of extracted thrombi from deep venous thrombosis and pulmonary embolism: Mechanisms and timing.

BACKGROUND: Mechanical thrombectomy is increasingly being used as an alternative to pharmacologic therapies for the treatment of patients with acute deep venous thrombosis (DVT) and pulmonary embolism (PE) and allows direct histopathologic comparison of thrombi extracted from living patients. We performed histopathologic analysis to thrombi extracted from cases of DVT and PE to gain insights into their relative cellular compositions. METHODS: Thrombus retrieved using a catheter-based thrombectomy system (ClotTriever for lower extremity DVT and FlowTriever for PE) from the 17 patients (7 DVT cases and 10 PE cases) were histologically evaluated. Histological features were used to estimate their age and pathological characteristics. RESULTS: The thrombus in all cases were composed of fibrin, platelets, red blood cells, and acute inflammatory cells. The weights of thrombus obtained from DVT versus PE cases were heavier (DVT 7.2 g (g) (5.6-10.2) vs. PE 4.8 g (3.6-6.8), p = .01). Overall thrombus healing (i.e., thrombus composed of smooth muscle cells, endothelial cells, and proteoglycans) was different between DVT and PE cases. 6/7 (86%) with features of late stage healing were from DVT cases while only three of ten (30%) were from PE cases while PE contained more acute thrombi with 7/10 (70%) stage 2 as compared 1/7 (14%) for DVT (p = .0498). CONCLUSION: This study is the first to directly compare the histology of extracted thrombus in DVT versus PE cases from patients with clinical events. Overall PE cases demonstrated significantly earlier stage thrombus with a larger component of red blood cells.