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1.
Platelets ; : 1-9, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31726921

RESUMO

Platelet decline is a feature of many acute viral infections, including cytomegalovirus (CMV) infection in humans and mice. Platelet sequestration in association with other cells, including endothelium and circulating leukocytes, can contribute to this decline and influence the immune response to and pathogenesis of viral infection. We sought to determine if platelet-endothelial associations (PEAs) contribute to platelet decline during acute murine CMV (mCMV) infection, and if these associations affect viral load and production. Male BALB/c mice were infected with mCMV (Smith strain), euthanized at timepoints throughout acute infection and compared to uninfected controls. An increase in PEA formation was confirmed in the salivary gland at all post-inoculation timepoints using immunohistochemistry for CD41+ platelets co-localizing with CD34+ vessels. Platelet depletion did not change amount of viral DNA or timecourse of infection, as measured by qPCR. However, platelet depletion reduced viral titer of mCMV in the salivary glands while undepleted controls demonstrated robust replication in the tissue by plaque assay. Thus, platelet associations with endothelium may enhance the ability of mCMV to replicate within the salivary gland. Further work is needed to determine the mechanisms behind this effect and if pharmacologic inhibition of PEAs may reduce CMV production in acutely infected patients.

2.
Mod Pathol ; 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31700162

RESUMO

Venous invasion is three times more common in pancreatic cancer than it is in other major cancers of the gastrointestinal tract, and venous invasion may explain why pancreatic cancer is so deadly. To characterize the patterns of venous invasion in pancreatic cancer, 52 thick slabs (up to 5 mm) of tissue were harvested from 52 surgically resected human ductal adenocarcinomas, cleared with a modified iDISCO method, and labeled with fluorescent-conjugated antibodies to cytokeratin 19, desmin, CD31, p53 and/or e-cadherin. Labeled three-dimensional (3D) pancreas cancer tissues were visualized with confocal laser scanning or light sheet microscopy. Multiple foci of venous and even arterial invasion were visualized. Venous invasion was detected more often in 3D (88%, 30/34 cases) than in conventional 2D slide evaluation (75%, 25/34 cases, P < 0.001). 3D visualization revealed pancreatic cancer cells crossing the walls of veins at multiple points, often at points where preexisting capillary structures bridge the blood vessels. The neoplastic cells often retained a ductal morphology (cohesive cells forming tubes) as they progressed from a stromal to intravenous location. Although immunolabeling with antibodies to e-cadherin revealed focal loss of expression at the leading edges of the cancers, the neoplastic cells within veins expressed e-cadherin and formed well-oriented glands. We conclude that venous invasion is almost universal in pancreatic cancer, suggesting that even surgically resectable PDAC has access to the venous spaces and thus the ability to disseminate widely. Furthermore, we observe that sustained epithelial-mesenchymal transition is not required for venous invasion in pancreatic cancer.

3.
Genome Med ; 11(1): 48, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31349857

RESUMO

BACKGROUND: Although mosaic variation has been known to cause disease for decades, high-throughput sequencing technologies with the analytical sensitivity to consistently detect variants at reduced allelic fractions have only recently emerged as routine clinical diagnostic tests. To date, few systematic analyses of mosaic variants detected by diagnostic exome sequencing for diverse clinical indications have been performed. METHODS: To investigate the frequency, type, allelic fraction, and phenotypic consequences of clinically relevant somatic mosaic single nucleotide variants (SNVs) and characteristics of the corresponding genes, we retrospectively queried reported mosaic variants from a cohort of ~ 12,000 samples submitted for clinical exome sequencing (ES) at Baylor Genetics. RESULTS: We found 120 mosaic variants involving 107 genes, including 80 mosaic SNVs in proband samples and 40 in parental/grandparental samples. Average mosaic alternate allele fraction (AAF) detected in autosomes and in X-linked disease genes in females was 18.2% compared with 34.8% in X-linked disease genes in males. Of these mosaic variants, 74 variants (61.7%) were classified as pathogenic or likely pathogenic and 46 (38.3%) as variants of uncertain significance. Mosaic variants occurred in disease genes associated with autosomal dominant (AD) or AD/autosomal recessive (AR) (67/120, 55.8%), X-linked (33/120, 27.5%), AD/somatic (10/120, 8.3%), and AR (8/120, 6.7%) inheritance. Of note, 1.7% (2/120) of variants were found in genes in which only somatic events have been described. Nine genes had recurrent mosaic events in unrelated individuals which accounted for 18.3% (22/120) of all detected mosaic variants in this study. The proband group was enriched for mosaicism affecting Ras signaling pathway genes. CONCLUSIONS: In sum, an estimated 1.5% of all molecular diagnoses made in this cohort could be attributed to a mosaic variant detected in the proband, while parental mosaicism was identified in 0.3% of families analyzed. As ES design favors breadth over depth of coverage, this estimate of the prevalence of mosaic variants likely represents an underestimate of the total number of clinically relevant mosaic variants in our cohort.

5.
Gastroenterology ; 157(4): 1123-1137.e22, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31175866

RESUMO

BACKGROUND & AIMS: Intraductal papillary mucinous neoplasms (IPMNs) are lesions that can progress to invasive pancreatic cancer and constitute an important system for studies of pancreatic tumorigenesis. We performed comprehensive genomic analyses of entire IPMNs to determine the diversity of somatic mutations in genes that promote tumorigenesis. METHODS: We microdissected neoplastic tissues from 6-24 regions each of 20 resected IPMNs, resulting in 227 neoplastic samples that were analyzed by capture-based targeted sequencing. Somatic mutations in genes associated with pancreatic tumorigenesis were assessed across entire IPMN lesions, and the resulting data were supported by evolutionary modeling, whole-exome sequencing, and in situ detection of mutations. RESULTS: We found a high prevalence of heterogeneity among mutations in IPMNs. Heterogeneity in mutations in KRAS and GNAS was significantly more prevalent in IPMNs with low-grade dysplasia than in IPMNs with high-grade dysplasia (P < .02). Whole-exome sequencing confirmed that IPMNs contained multiple independent clones, each with distinct mutations, as originally indicated by targeted sequencing and evolutionary modeling. We also found evidence for convergent evolution of mutations in RNF43 and TP53, which are acquired during later stages of tumorigenesis. CONCLUSIONS: In an analysis of the heterogeneity of mutations throughout IPMNs, we found that early-stage IPMNs contain multiple independent clones, each with distinct mutations, indicating their polyclonal origin. These findings challenge the model in which pancreatic neoplasms arise from a single clone. Increasing our understanding of the mechanisms of IPMN polyclonality could lead to strategies to identify patients at increased risk for pancreatic cancer.


Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Mutação , Neoplasias Intraductais Pancreáticas/genética , Neoplasias Pancreáticas/genética , Idoso , Idoso de 80 Anos ou mais , Transformação Celular Neoplásica/patologia , Cromograninas/genética , Evolução Clonal , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Evolução Molecular , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Estadiamento de Neoplasias , Proteínas Oncogênicas/genética , Neoplasias Intraductais Pancreáticas/patologia , Neoplasias Pancreáticas/patologia , Fenótipo , Proteínas Proto-Oncogênicas p21(ras)/genética , Estudos Retrospectivos
6.
Genome Med ; 11(1): 30, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101064

RESUMO

BACKGROUND: Exome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES. METHODS: In a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array. RESULTS: The QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities. CONCLUSIONS: Our clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations.

7.
Genome Med ; 10(1): 74, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266093

RESUMO

BACKGROUND: Exome sequencing is now being incorporated into clinical care for pediatric and adult populations, but its integration into prenatal diagnosis has been more limited. One reason for this is the paucity of information about the clinical utility of exome sequencing in the prenatal setting. METHODS: We retrospectively reviewed indications, results, time to results (turnaround time, TAT), and impact of exome results for 146 consecutive "fetal exomes" performed in a clinical diagnostic laboratory between March 2012 and November 2017. We define a fetal exome as one performed on a sample obtained from a fetus or a product of conception with at least one structural anomaly detected by prenatal imaging or autopsy. Statistical comparisons were performed using Fisher's exact test. RESULTS: Prenatal exome yielded an overall molecular diagnostic rate of 32% (n = 46/146). Of the 46 molecular diagnoses, 50% were autosomal dominant disorders (n = 23/46), 41% were autosomal recessive disorders (n = 19/46), and 9% were X-linked disorders (n = 4/46). The molecular diagnostic rate was highest for fetuses with anomalies affecting multiple organ systems and for fetuses with craniofacial anomalies. Out of 146 cases, a prenatal trio exome option designed for ongoing pregnancies was performed on 62 fetal specimens, resulting in a diagnostic yield of 35% with an average TAT of 14 days for initial reporting (excluding tissue culture time). The molecular diagnoses led to refined recurrence risk estimates, altered medical management, and informed reproductive planning for families. CONCLUSION: Exome sequencing is a useful diagnostic tool when fetal structural anomalies suggest a genetic etiology, but other standard prenatal genetic tests did not provide a diagnosis.

8.
Genet Med ; 2018 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-30214068

RESUMO

PURPOSE: Clinical laboratories performing exome or genome sequencing (ES/GS) are familiar with the challenges associated with proper consenting for and reporting of medically actionable secondary findings based on recommendations from the American College of Medical Genetics and Genomics (ACMG). Misattributed parentage is another type of unanticipated finding a laboratory may encounter during family-based ES/GS; however, there are currently no professional recommendations related to the proper consenting for and reporting of misattributed parentage encountered during ES/GS. METHODS: We surveyed 10 clinical laboratories offering family-based ES/GS regarding their consent language, discovery, and reporting of misattributed parentage. RESULTS: Many laboratories have already developed their own practices/policies for these issues, which do not necessarily agree with those from other labs. CONCLUSION: There are several other possibilities besides true misattributed parentage that could result in similar laboratory findings, and laboratories often feel they lack sufficient information to make formal conclusions on a report regarding the true genetic relatedness of the submitted samples. However, understanding the genetic relatedness (or lack thereof) of the samples submitted for family-based ES/GS has medical relevance. Therefore, professional recommendations for the appropriate handling of suspected misattributed parentage encountered during ES/GS are needed to help standardize current clinical laboratory practices.

9.
Am J Med Genet A ; 176(4): 973-979, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29423971

RESUMO

SHANK3 encodes for a scaffolding protein that links neurotransmitter receptors to the cytoskeleton and is enriched in postsynaptic densities of excitatory synapses. Deletions or mutations in one copy of the SHANK3 gene cause Phelan-McDermid syndrome, also called 22q13.3 deletion syndrome, a neurodevelopmental disorder with common features including global developmental delay, absent to severely impaired language, autistic behavior, and minor dysmorphic features. By whole exome sequencing, we identified two de novo novel variants including one frameshift pathogenic variant and one missense variant of unknown significance in a 14-year-old boy with delayed motor milestones, delayed language acquisition, autism, intellectual disability, ataxia, progressively worsening spasticity of the lower extremities, dysmorphic features, short stature, microcephaly, failure to thrive, chronic constipation, intrauterine growth restriction, and bilateral inguinal hernias. Both changes are within the CpG island in exon 21, separated by a 375 bp sequence. Next generation sequencing of PCR products revealed that the two variants are most frequently associated with each other. Sanger sequencing of the cloned PCR products further confirmed that both changes were on a single allele. The clinical presentation in this individual is consistent with other patients with a truncating mutation in exon 21, suggesting that the missense change contributes none or minimally to the phenotypes. This is the first report of two de novo mutations in one SHANK3 allele.

10.
Cell Rep ; 21(7): 1896-1909, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29141221

RESUMO

Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy.


Assuntos
Antineoplásicos/farmacologia , Carcinoma/patologia , Cisplatino/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Teratoma/patologia , Neoplasias Testiculares/patologia , Animais , Apoptose , Carcinoma/metabolismo , Transformação Celular Neoplásica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Teratoma/metabolismo , Neoplasias Testiculares/metabolismo
11.
JAMA Pediatr ; 171(12): e173438, 2017 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-28973083

RESUMO

Importance: While congenital malformations and genetic diseases are a leading cause of early infant death, to our knowledge, the contribution of single-gene disorders in this group is undetermined. Objective: To determine the diagnostic yield and use of clinical exome sequencing in critically ill infants. Design, Setting, and Participants: Clinical exome sequencing was performed for 278 unrelated infants within the first 100 days of life who were admitted to Texas Children's Hospital in Houston, Texas, during a 5-year period between December 2011 and January 2017. Exome sequencing types included proband exome, trio exome, and critical trio exome, a rapid genomic assay for seriously ill infants. Main Outcomes and Measures: Indications for testing, diagnostic yield of clinical exome sequencing, turnaround time, molecular findings, patient age at diagnosis, and effect on medical management among a group of critically ill infants who were suspected to have genetic disorders. Results: The mean (SEM) age for infants participating in the study was 28.5 (1.7) days; of these, the mean (SEM) age was 29.0 (2.2) days for infants undergoing proband exome sequencing, 31.5 (3.9) days for trio exome, and 22.7 (3.9) days for critical trio exome. Clinical indications for exome sequencing included a range of medical concerns. Overall, a molecular diagnosis was achieved in 102 infants (36.7%) by clinical exome sequencing, with relatively low yield for cardiovascular abnormalities. The diagnosis affected medical management for 53 infants (52.0%) and had a substantial effect on informed redirection of care, initiation of new subspecialist care, medication/dietary modifications, and furthering life-saving procedures in select patients. Critical trio exome sequencing revealed a molecular diagnosis in 32 of 63 infants (50.8%) at a mean (SEM) of 33.1 (5.6) days of life with a mean (SEM) turnaround time of 13.0 (0.4) days. Clinical care was altered by the diagnosis in 23 of 32 patients (71.9%). The diagnostic yield, patient age at diagnosis, and medical effect in the group that underwent critical trio exome sequencing were significantly different compared with the group who underwent regular exome testing. For deceased infants (n = 81), genetic disorders were molecularly diagnosed in 39 (48.1%) by exome sequencing, with implications for recurrence risk counseling. Conclusions and Relevance: Exome sequencing is a powerful tool for the diagnostic evaluation of critically ill infants with suspected monogenic disorders in the neonatal and pediatric intensive care units and its use has a notable effect on clinical decision making.


Assuntos
Doenças Genéticas Inatas/diagnóstico , Unidades de Terapia Intensiva Pediátrica , Sequenciamento Completo do Exoma/métodos , Adulto , Cuidados Críticos/métodos , Gerenciamento Clínico , Exoma , Aconselhamento Genético/métodos , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Humanos , Lactente , Cuidado do Lactente/métodos , Recém-Nascido , Tempo de Internação/estatística & dados numéricos , Estudos Retrospectivos , Texas
12.
Am J Obstet Gynecol ; 217(6): 691.e1-691.e6, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29032050

RESUMO

BACKGROUND: Since its debut in 2011, cell-free fetal DNA screening has undergone rapid expansion with respect to both utilization and coverage. However, conclusive data regarding the clinical validity and utility of this screening tool, both for the originally included common autosomal and sex-chromosomal aneuploidies as well as the more recently added chromosomal microdeletion syndromes, have lagged behind. Thus, there is a continued need to educate clinicians and patients about the current benefits and limitations of this screening tool to inform pre- and posttest counseling, pre/perinatal decision making, and medical risk assessment/management. OBJECTIVE: The objective of this study was to determine the positive predictive value and false-positive rates for different chromosomal abnormalities identified by cell-free fetal DNA screening using a large data set of diagnostic testing results on invasive samples submitted to the laboratory for confirmatory studies. STUDY DESIGN: We tested 712 patient samples sent to our laboratory to confirm a cell-free fetal DNA screening result, indicating high risk for a chromosome abnormality. We compiled data from all cases in which the indication for confirmatory testing was a positive cell-free fetal DNA screen, including the common trisomies, sex chromosomal aneuploidies, microdeletion syndromes, and other large genome-wide copy number abnormalities. Testing modalities included fluorescence in situ hybridization, G-banded karyotype, and/or chromosomal microarray analysis performed on chorionic villus samples, amniotic fluid, or postnatally obtained blood samples. Positive predictive values and false-positive rates were calculated from tabulated data. RESULTS: The positive predictive values for trisomy 13, 18, and 21 were consistent with previous reports at 45%, 76%, and 84%, respectively. For the microdeletion syndrome regions, positive predictive values ranged from 0% for detection of Cri-du-Chat syndrome and Prader-Willi/Angelman syndrome to 14% for 1p36 deletion syndrome and 21% for 22q11.2 deletion syndrome. Detection of sex chromosomal aneuploidies had positive predictive values of 26% for monosomy X, 50% for 47,XXX, and 86% for 47,XXY. CONCLUSION: The positive predictive values for detection of common autosomal and sex chromosomal aneuploidies by cell-free fetal DNA screening were comparable with other studies. Identification of microdeletions was associated with lower positive predictive values and higher false-positive rates, likely because of the low prevalence of the individual targeted microdeletion syndromes in the general population. Although the obtained positive predictive values compare favorably with those seen in traditional screening approaches for common aneuploidies, they highlight the importance of educating clinicians and patients on the limitations of cell-free fetal DNA screening tests. Improvement of the cell-free fetal DNA screening technology and continued monitoring of its performance after introduction into clinical practice will be important to fully establish its clinical utility. Nonetheless, our data provide valuable information that may aid result interpretation, patient counseling, and clinical decision making/management.


Assuntos
Ácidos Nucleicos Livres/sangue , Transtornos Cromossômicos/sangue , Amniocentese , Síndrome de Angelman/sangue , Síndrome de Angelman/diagnóstico , Síndrome de Angelman/genética , Amostra da Vilosidade Coriônica , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Cromossomos Humanos X/genética , Síndrome do Miado do Gato/sangue , Síndrome do Miado do Gato/diagnóstico , Síndrome do Miado do Gato/genética , Síndrome de Down/sangue , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Síndrome de Klinefelter/sangue , Síndrome de Klinefelter/diagnóstico , Síndrome de Klinefelter/genética , Análise em Microsséries , Síndrome de Prader-Willi/sangue , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , Valor Preditivo dos Testes , Gravidez , Diagnóstico Pré-Natal , Aberrações dos Cromossomos Sexuais , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/sangue , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/diagnóstico , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/genética , Trissomia/diagnóstico , Trissomia/genética , Síndrome da Trissomia do Cromossomo 13/sangue , Síndrome da Trissomia do Cromossomo 13/diagnóstico , Síndrome da Trissomia do Cromossomo 13/genética , Síndrome da Trissomía do Cromossomo 18/sangue , Síndrome da Trissomía do Cromossomo 18/diagnóstico , Síndrome da Trissomía do Cromossomo 18/genética , Síndrome de Turner/sangue , Síndrome de Turner/diagnóstico , Síndrome de Turner/genética
13.
Virology ; 508: 134-149, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28531865

RESUMO

Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that is partially prevented by treatment with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist (Potter et al., J Neurovirol 21:159, 2015). To determine the mechanism(s) of protection, lymph node and central nervous system (CNS) tissues from SINV-infected mice treated daily for 1 week with low (0.3mg/kg) or high (0.6mg/kg) dose DON were examined. DON treatment suppressed lymphocyte proliferation in cervical lymph nodes resulting in reduced CNS immune cell infiltration, inflammation, and cell death compared to untreated SINV-infected mice. Production of SINV-specific antibody and interferon-gamma were also impaired by DON treatment with a delay in virus clearance. Cessation of treatment allowed activation of the antiviral immune response and viral clearance, but revived CNS pathology, demonstrating the ability of the immune response to mediate both CNS damage and virus clearance.


Assuntos
Infecções por Alphavirus/tratamento farmacológico , Infecções por Alphavirus/imunologia , Antivirais/administração & dosagem , Diazo-Oxo-Norleucina/administração & dosagem , Encefalomielite/tratamento farmacológico , Encefalomielite/imunologia , Glutamina/antagonistas & inibidores , Vírus Sindbis/fisiologia , Infecções por Alphavirus/patologia , Infecções por Alphavirus/virologia , Animais , Encefalomielite/patologia , Encefalomielite/virologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vírus Sindbis/efeitos dos fármacos
14.
Am J Hum Genet ; 100(4): 676-688, 2017 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-28343629

RESUMO

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.


Assuntos
Anormalidades Múltiplas/genética , Endopeptidases/genética , Deficiência Intelectual/genética , Adolescente , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Deleção de Genes , Humanos , Masculino , Camundongos , Linhagem , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Convulsões/genética
15.
Am J Hum Genet ; 99(4): 886-893, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27616478

RESUMO

Disruption of the establishment of left-right (L-R) asymmetry leads to situs anomalies ranging from situs inversus totalis (SIT) to situs ambiguus (heterotaxy). The genetic causes of laterality defects in humans are highly heterogeneous. Via whole-exome sequencing (WES), we identified homozygous mutations in PKD1L1 from three affected individuals in two unrelated families. PKD1L1 encodes a polycystin-1-like protein and its loss of function is known to cause laterality defects in mouse and medaka fish models. Family 1 had one fetus and one deceased child with heterotaxy and complex congenital heart malformations. WES identified a homozygous splicing mutation, c.6473+2_6473+3delTG, which disrupts the invariant splice donor site in intron 42, in both affected individuals. In the second family, a homozygous c.5072G>C (p.Cys1691Ser) missense mutation was detected in an individual with SIT and congenital heart disease. The p.Cys1691Ser substitution affects a highly conserved cysteine residue and is predicted by molecular modeling to disrupt a disulfide bridge essential for the proper folding of the G protein-coupled receptor proteolytic site (GPS) motif. Damaging effects associated with substitutions of this conserved cysteine residue in the GPS motif have also been reported in other genes, namely GPR56, BAI3, and PKD1 in human and lat-1 in C. elegans, further supporting the likely pathogenicity of p.Cys1691Ser in PKD1L1. The identification of bi-allelic PKD1L1 mutations recapitulates previous findings regarding phenotypic consequences of loss of function of the orthologous genes in mice and medaka fish and further expands our understanding of genetic contributions to laterality defects in humans.


Assuntos
Alelos , Lateralidade Funcional/genética , Proteínas de Membrana/genética , Mutação , Situs Inversus/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Cisteína/genética , Exoma/genética , Feminino , Doenças Fetais/genética , Cardiopatias Congênitas/genética , Síndrome de Heterotaxia , Homozigoto , Humanos , Recém-Nascido , Íntrons/genética , Masculino , Proteínas de Membrana/química , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Mutação de Sentido Incorreto , Oryzias/genética , Linhagem , Processamento de RNA/genética
16.
Am J Hum Genet ; 99(3): 720-727, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27545676

RESUMO

SON is a key component of the spliceosomal complex and a critical mediator of constitutive and alternative splicing. Additionally, SON has been shown to influence cell-cycle progression, genomic integrity, and maintenance of pluripotency in stem cell populations. The clear functional relevance of SON in coordinating essential cellular processes and its presence in diverse human tissues suggests that intact SON might be crucial for normal growth and development. However, the phenotypic effects of deleterious germline variants in SON have not been clearly defined. Herein, we describe seven unrelated individuals with de novo variants in SON and propose that deleterious variants in SON are associated with a severe multisystem disorder characterized by developmental delay, persistent feeding difficulties, and congenital malformations, including brain anomalies.


Assuntos
Anormalidades Congênitas/genética , Proteínas de Ligação a DNA/genética , Deficiências do Desenvolvimento/genética , Insuficiência de Crescimento/genética , Deficiência Intelectual/genética , Antígenos de Histocompatibilidade Menor/genética , Deleção de Sequência/genética , Adolescente , Encéfalo/anormalidades , Criança , Pré-Escolar , Proteínas de Ligação a DNA/química , Exoma/genética , Feminino , Humanos , Masculino , Antígenos de Histocompatibilidade Menor/química , Linhagem , Adulto Jovem
17.
Am J Med Genet A ; 170A(4): 891-5, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26686323

RESUMO

Filamin A (FLNA) is known to be involved in intracellular actin binding, cell migration, scaffolding, and signaling. We report a novel X-linked syndrome characterized by cardiac valvular disease, keloid scarring and reduced joint mobility in male second cousins due to a previously unreported mutation in FLNA. Whole exome sequencing was performed using standard methods and segregation analysis was performed in affected and non-affected family members. A novel hemizygous c.4726G>A (p.G1576R) mutation in FLNA was detected. Segregation analysis performed on multiple maternal family members showed c.4726G>A (p.G1576R) segregated with disease in an X-linked inheritance pattern. The findings in these cases are distinct from previously described FLNA related disorders by virtue of decreased joint mobility and spontaneous keloid scarring. They occur in association with a novel mutation and represent a novel genetic syndrome.


Assuntos
Substituição de Aminoácidos , Filaminas/genética , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação , Fenótipo , Adulto , Alelos , Códon , Facies , Estudos de Associação Genética , Genótipo , Humanos , Queloide/patologia , Masculino , Linhagem , Síndrome , Adulto Jovem
18.
J Genet Couns ; 24(4): 654-62, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25403901

RESUMO

The purpose of this study was to investigate how the American College of Medical Genetics and Genomics (ACMG) March 2013 recommendations for reporting incidental findings (IFs) have influenced current practices of genetic counselors involved in utilizing whole exome sequencing (WES) for clinical diagnosis. An online survey was sent to all members of the National Society of Genetic Counselors; members were eligible to participate if they currently offered WES for clinical diagnosis. Forty-six respondents completed the survey of whom 34 were in practice prior to the March 2013 ACMG recommendations. Half of respondents (N = 19, 54.9 %) in practice prior to March 2013 reported that the ACMG recommendations have had a significant impact on the content of their counseling sessions. Approximately half of respondents (N = 21, 45.5 %) report all IFs, regardless of patient age, while one third (N = 14, 30.4 %) consider factors such as age and parent preference in reporting IFs. Approximately 40 % (N = 18) of respondents reported that the testing laboratory's policy for returning IFs has an influence on their choice of laboratory; of those, 72.2 % (N = 13) reported that the option to opt out of receiving reports of IFs has a significant influence on their choice of laboratory. A majority of respondents (N = 43, 93.5 %) found that most patients want to receive reports of IFs. However, respondents report there are patients who wish to decline receiving this information. This study querying genetic counselors identified benefits and challenges that the 2013 ACMG recommendations elicited. Some challenges, such as not having the option to opt out of IFs, have been addressed by the ACMG's most recent updates to their recommendations. Further investigation into larger and more inclusive provider populations as well as patient populations will be valuable for the ongoing discussion surrounding IFs in WES.


Assuntos
Exoma/genética , Aconselhamento Genético/métodos , Estudo de Associação Genômica Ampla , Fidelidade a Diretrizes , Achados Incidentais , Registros Médicos Orientados a Problemas , Análise de Sequência , Adulto , Atitude do Pessoal de Saúde , Criança , Feminino , Testes Genéticos , Humanos , Recém-Nascido , Masculino , Gravidez , Inquéritos e Questionários
19.
JAMA ; 312(18): 1870-9, 2014 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-25326635

RESUMO

IMPORTANCE: Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders. OBJECTIVE: To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome. DESIGN, SETTING, AND PATIENTS: Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patient's physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay. MAIN OUTCOMES AND MEASURES: Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings. RESULTS: A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%-31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%-27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%-47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%-25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics. CONCLUSIONS AND RELEVANCE: Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients.


Assuntos
Exoma , Doenças Genéticas Inatas/diagnóstico , Técnicas de Diagnóstico Molecular , Análise de Sequência de DNA/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Feto , Testes Genéticos , Genômica , Humanos , Achados Incidentais , Lactente , Recém-Nascido , Masculino , Mutação , Fenótipo , Encaminhamento e Consulta
20.
J Genet Couns ; 23(4): 496-505, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24671342

RESUMO

In recent years, new sequencing technologies known as next generation sequencing (NGS) have provided scientists the ability to rapidly sequence all known coding as well as non-coding sequences in the human genome. As the two emerging approaches, whole exome (WES) and whole genome (WGS) sequencing, have started to be integrated in the clinical arena, we sought to survey health care professionals who are likely to be involved in the implementation process now and/or in the future (e.g., genetic counselors, geneticists and nurse practitioners). Two hundred twenty-one genetic counselors- one third of whom currently offer WES/WGS-participated in an anonymous online survey. The aims of the survey were first, to identify barriers to the implementation of WES/WGS, as perceived by survey participants; second, to provide the first systematic report of current practices regarding the integration of WES/WGS in clinic and/or research across the US and Canada and to illuminate the roles and challenges of genetic counselors participating in this process; and third to evaluate the impact of WES/WGS on patient care. Our results showed that genetic counseling practices with respect to WES/WGS are consistent with the criteria set forth in the ACMG 2012 policy statement, which highlights indications for testing, reporting, and pre/post test considerations. Our respondents described challenges related to offering WES/WGS, which included billing issues, the duration and content of the consent process, result interpretation and disclosure of incidental findings and variants of unknown significance. In addition, respondents indicated that specialty area (i.e., prenatal and cancer), lack of clinical utility of WES/WGS and concerns about interpretation of test results were factors that prevented them from offering this technology to patients. Finally, study participants identified the aspects of their professional training which have been most beneficial in aiding with the integration of WES/WGS into the clinical setting (molecular/clinical genetics, counseling and bioethics) and suggested that counseling aids (to assist them when explaining aspects of these tests to patients) and webinars focused on WES/WGS (for genetic counselors and other health care professionals) would be useful educational tools. Future research should permit us to further enhance our knowledge of pitfalls and benefits associated with the introduction of these powerful technologies in patient care and to further explore the roles and opportunities for genetic counselors in this rapidly evolving field.


Assuntos
Aconselhamento Genético/psicologia , Genoma Humano , Atitude do Pessoal de Saúde , Humanos
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