RESUMO
Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder featuring chronic lymphadenopathy, splenomegaly, cytopenias, and increased lymphoma risk. Differentiating ALPS from immunodeficiencies with overlapping symptoms is challenging. This study evaluated the performance and the diagnostic yield of a 15-gene NGS panel for ALPS at Cincinnati Children's Hospital Medical Center. Samples from 802 patients submitted for ALPS NGS panel were studied between May 2014 and January 2023. A total of 62 patients (7.7%) had a definite diagnosis: 52/62 cases (84%) showed 37 unique pathogenic/likely pathogenic germline FAS variants supporting ALPS diagnosis (6.5%, 52/802). The ALPS diagnostic yield increased to 30% in patients who additionally fulfilled abnormal ALPS immunology findings criteria. 17/37 (46%) diagnostic FAS variants were novel variants reported for the first time in ALPS. 10/802 cases (1.2%) showed diagnostic findings in five genes (ADA2, CTLA4, KRAS, MAGT1, NRAS) which are related to autoimmune lymphoproliferative immunodeficiency (ALPID). Family studies enabled the reclassification of variants of unknown significance (VUS) and also the identification of at-risk family members of FAS-positive patients, which helped in the follow-up diagnosis and treatment. Alongside family studies, complete clinical phenotypes and abnormal ALPS immunology and Fas-mediated apoptosis results helped clarify uncertain genetic findings. This study describes the largest cohort of genetic testing for suspected ALPS in North America and highlights the effectiveness of the ALPS NGS panel in distinguishing ALPS from non-ALPS immunodeficiencies. More comprehensive assessment from exome or genome sequencing could be considered for undefined ALPS-U patients or non-ALPS immunodeficiencies after weighing cost, completeness, and timeliness of different genetic testing options.
Assuntos
Síndrome Linfoproliferativa Autoimune , Testes Genéticos , Humanos , Síndrome Linfoproliferativa Autoimune/diagnóstico , Síndrome Linfoproliferativa Autoimune/genética , Testes Genéticos/métodos , Feminino , Masculino , Criança , Pré-Escolar , Lactente , Adolescente , Receptor fas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Predisposição Genética para Doença , Hospitais Pediátricos , Mutação/genéticaRESUMO
OBJECTIVE: To investigate the prevalence of genetic disease and its economic impact in a level IV neonatal intensive care unit (NICU) by identifying and describing diseases diagnosed, genetic testing methodologies used, timing of diagnosis, length of NICU stay, and charges for NICU care. STUDY DESIGN: A retrospective chart review of patients admitted to a level IV NICU from 2013 to 2014 (n = 1327) was undertaken and data collected up to 2 years of age from the electronic medical record. RESULTS: In total, 117 patients (9%) received 120 genetic diagnoses using a variety of methodologies. A significant minority of diagnoses, 36%, were made after NICU discharge and 41% were made after 28 days of age. Patients receiving a genetic diagnosis had significantly longer mean lengths of stay (46 days vs 29.1 days; P < .01) and costlier mean charges ($598 712 vs $352 102; P < .01) for their NICU care. The NICU stay charge difference to care for a newborn with a genetic condition was on average $246â610 in excess of that for a patient without a genetic diagnosis, resulting in more than $28â000â000 in excess charges to care for all patients with genetic conditions in a single NICU over a 2-year period. CONCLUSIONS: Given the high prevalence of genetic disease in this population and the documented higher cost of care, shortening the time to diagnosis and targeting therapeutic interventions for this population could make a significant impact on neonatal care in level IV NICUs.
Assuntos
Doenças Genéticas Inatas/economia , Doenças Genéticas Inatas/genética , Testes Genéticos/economia , Testes Genéticos/métodos , Unidades de Terapia Intensiva Neonatal , Terapia Intensiva Neonatal/economia , Metilação de DNA , Registros Eletrônicos de Saúde , Exoma , Feminino , Doenças Genéticas Inatas/diagnóstico , Humanos , Hibridização in Situ Fluorescente , Lactente , Mortalidade Infantil , Recém-Nascido , Tempo de Internação , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Alta do Paciente , Prevalência , Estudos Retrospectivos , Análise de Sequência de DNARESUMO
Variable lung disease was documented in 2 infants with heterozygous TBX4 mutations; their clinical presentations, pathology, and outcomes were distinct. These findings demonstrate that TBX4 gene mutations are associated with neonatal respiratory failure and highlight the wide spectrum of clinicopathological outcomes that have implications for patient diagnosis and management.
Assuntos
Mutação/genética , Insuficiência Respiratória/genética , Insuficiência Respiratória/patologia , Proteínas com Domínio T/genética , Feminino , Humanos , Recém-Nascido , MasculinoRESUMO
BACKGROUND: European studies identified the C9orf72 repeat expansion as the most frequent genetic alteration in patients with Huntington disease (HD)-like phenotypes but negative HD genetic testing. OBJECTIVE: To investigate C9orf72 repeat expansion frequency in individuals tested for HD in a North American tertiary referral laboratory. METHODS: Three hundred and seventy-three cases (115 positive and 258 negative for HD) were evaluated by genotyping PCR, with follow-up Southern blot and 5' repeat methylation status assessment by combined repeat-primed and methylation-specific PCR in a subset. RESULTS: Three cases (all HD-negative) tested positive: 2 had > 2,000 repeats and were methylated, 1 had 80-100 repeats and was unmethylated. Two cases (1 HD-positive and 1 HD-negative) had intermediate alleles (20-29 repeats) and were unmethylated. The remaining 368 cases were negative (< 20 repeats). C9orf72 repeat expansion was absent in patients with HD and was identified in a small subset (1.2%) of patients with negative HD genetic testing. CONCLUSION: These findings suggest that C9orf72 repeat expansion does not coexist with HTT repeat expansion and that C9orf72 repeat expansion testing is unnecessary for patients with HD. In addition, C9orf72 evaluation may be considered for individuals negative for HD genetic testing. Similar to in previous studies, methylation of C9orf72 repeat expansion was limited to large expansions.
Assuntos
Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Doença de Huntington/genética , Mutação/genética , Adulto , Idoso , Esclerose Lateral Amiotrófica/genética , Feminino , Testes Genéticos/métodos , Humanos , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
Deletion of 15q11.2-q13 results in either Prader-Willi syndrome (PWS) or Angelman syndrome (AS) depending on the parent of origin. Duplication of the PWS/AS critical region (PWASCR) has also been reported in association with developmental delay and autism, and it has been shown that they also show a parent-of-origin effect. It is generally accepted that maternal duplications are pathogenic. However, there is conflicting evidence as to the pathogenicity of paternal duplications. We have identified 35 patients with gain of the PWASCR using array comparative genomic hybridization. Methylation testing was performed to determine parent of origin of the extra copies. Of the 35 cases, 22 had a supernumerary marker chromosome 15 (SMC15), 12 had a tandem duplication, and 1 had a tandem triplication. Only one patient had a paternal duplication; this patient does not have features typical of patients with maternal duplication of the PWASCR. Three of the mothers had a tandem duplication (two were paternal and one was maternal origin). While one of the two mothers with paternal duplication was noted not to have autism, the other was noted to have learning disability and depression. Based on our data, we conclude that SMC15 are almost exclusively maternal in origin and result in an abnormal phenotype. Tandem duplications/triplications are generally of maternal origin when ascertained on the basis of abnormal phenotype; however, tandem duplications of paternal origin have also been identified. Therefore, we suggest that methylation testing be performed for cases of tandem duplications/triplications since the pathogenicity of paternal gains is uncertain.
Assuntos
Síndrome de Angelman/genética , Metilação de DNA/genética , Dosagem de Genes/genética , Duplicação Gênica/genética , Síndrome de Prader-Willi/genética , Adolescente , Adulto , Criança , Pré-Escolar , Duplicação Cromossômica/genética , Cromossomos Humanos Par 15/genética , Deficiências do Desenvolvimento/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pais , Fenótipo , Deleção de Sequência/genética , Adulto JovemRESUMO
Hb Memphis [α23(B4)GluâGln; HBA2: c.70G > C (or HBA1)] is a stable hemoglobin (Hb) variant caused by a substitution of glutamine for glutamic acid at residue 23 of the α2- or α1-globin chain. Heterozygous Hb Memphis has no known clinical or hematological effect, and all prior reports have resulted from observations in persons of African descent with sickle cell disease and an unusually mild clinical course. Family studies suggest that Hb Memphis may modulate sickling. Only brief characterizations of Hb Memphis trait in the absence of Hb S are present in the current literature. We report isolated Hb Memphis trait in Turkish individuals in whom the initial laboratory incorrectly identified the α variant as Q-Thailand [α74(EF3)AspâHis; HBA1: c.223G > C]. In one case, a heterozygous -3.7 kb α gene deletion was also present, which increased the variant Hb level to a percentage similar to that of the more common Hb Q-Thailand, which may have led to the misidentification. Herein, we discuss the characterization and comparison of these variants and underscore the necessity of confirming characterization by more than one method prior to assigning Hb variant identification.
Assuntos
Hemoglobina A2/genética , Hemoglobinas Anormais/genética , Mutação de Sentido Incorreto , Cátions , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Ácido Glutâmico/genética , Glutamina/genética , Hemoglobinas Glicadas/genética , Hemoglobinas Glicadas/metabolismo , Hemoglobina A2/metabolismo , Hemoglobinas Anormais/metabolismo , Humanos , Lactente , Troca Iônica , Masculino , Mutação Puntual , Tailândia , TurquiaRESUMO
Hyperunstable hemoglobinopathy (HUH) [dominantly inherited ß-thalassemia (ß-thal)] is a relatively rare form of congenital hemolytic anemia in which mutations occur in the genes encoding for α and ß chains, or both chains of the hemoglobin (Hb) molecule. We describe two Hispanic adolescents with a new unstable Hb variant (HBB: c.348_349delinsG; p.His117IlefsX42), resulting from a frameshift mutation at codons 115/116 of the ß-globin gene. Both patients also have a 3.7 kb deletion on one α gene, leading to a decreased imbalance between α and ß chain formation, and subsequently a milder phenotype than that seen in other hyperunstable Hb variants.
Assuntos
Hemoglobinopatias/sangue , Hemoglobinopatias/genética , Hemoglobinas Anormais/genética , Adolescente , Substituição de Aminoácidos , Códon , Eritrócitos Anormais , Corpos de Heinz , Hemoglobinopatias/diagnóstico , Hemoglobinas Anormais/metabolismo , Heterozigoto , Humanos , Masculino , Mutação , Estabilidade Proteica , Irmãos , alfa-Globinas/genética , Globinas beta/genéticaRESUMO
PURPOSE: We describe the implementation of CYP2D6-focused pharmacogenetic testing to guide opioid prescribing in a quaternary care, nonprofit pediatric academic medical center. SUMMARY: Children are often prescribed oral opioids after surgeries, for cancer pain, and occasionally for chronic pain. In 2004, Cincinnati Children's Hospital Medical Center implemented pharmacogenetic testing for CYP2D6 metabolism phenotype to inform codeine prescribing. The test and reports were updated to align with changes over time in the testing platform, the interpretation of genotype to phenotype, the electronic health record, and Food and Drug Administration (FDA) guidance. The use of the test increased when a research project required testing and decreased as prescribing of oxycodone increased due to FDA warnings about codeine. Education about the opioid-focused pharmacogenetic test was provided to prescribers (eg, the pain and sickle cell teams) as well as patients and families. Education and electronic health record capability increased provider compliance with genotype-guided postsurgical prescribing of oxycodone, although there was a perceived lack of utility for oxycodone prescribing. CONCLUSION: The implementation of pharmacogenetic testing to inform opioid prescribing for children has evolved with accumulating evidence and guidelines, requiring changes in reporting of results and recommendations.
Assuntos
Analgésicos Opioides , Dor Crônica , Humanos , Analgésicos Opioides/efeitos adversos , Oxicodona , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Farmacogenética/métodos , Padrões de Prática Médica , Codeína/efeitos adversos , Dor Crônica/tratamento farmacológicoRESUMO
Our institution developed and continuously improved a Neurodevelopmental Reflex (NDR) algorithm to help physicians with genetic test ordering for neurodevelopmental disorders (NDDs). To assess its performance, we performed a retrospective study of 511 patients tested through NDR from 2018 to 2019. SNP Microarray identified pathogenic/likely pathogenic copy number variations in 27/511 cases (5.28%). Among the 484 patients tested for Fragile X FMR1 CGG repeats, a diagnosis (0.20%) was established for one male mosaic for a full mutation, a premutation, and a one-CGG allele. Within the 101 normocephalic female patients tested for MECP2, two patients were found to carry pathogenic variants (1.98%). This retrospective study suggested the NDR algorithm effectively established diagnoses for patients with NDDs with a yield of 5.87%.
Assuntos
Transtorno do Espectro Autista , Síndrome do Cromossomo X Frágil , Transtornos do Neurodesenvolvimento , Transtorno do Espectro Autista/diagnóstico , Criança , Variações do Número de Cópias de DNA , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/genética , Testes Genéticos , Hospitais , Humanos , Masculino , Mutação , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética , Estudos Retrospectivos , Expansão das Repetições de TrinucleotídeosRESUMO
PURPOSE: Approximately 8% of couples attempting to conceive are infertile and male infertility accounts for approximately 50% of infertility among couples. Up to 25% of males with non-obstructive infertility have chromosomal abnormalities and/or microdeletions of the long arm of the Y-chromosome. These are detected by conventional chromosome and Y-microdeletion analysis. In this study, we reviewed the results of testing performed in the Mayo Clinic Cytogenetics and Molecular Genetics Laboratories and compared our findings with previously published reports. METHODS: This study includes 2,242 chromosome studies from males ≥18 years of age referred for infertility between 1989 and 2000 and 2,749 Y-deletion molecular studies performed between 2002 and 2009. RESULTS: 14.3% of infertile males tested by karyotyping had abnormalities identified. These include: (258) 47,XXY and variants consistent with Klinefelter syndrome, (3) combined 47,XXY and balanced autosomal rearrangements, (9) 47,XYY, (9) Y-deletions, (7) 46,XX males, (32) balanced rearrangements, and (1) unbalanced rearrangement. 3.6% of males tested for Y-microdeletion analysis had abnormalities identified, 90% of which included a deletion of the AZFc region. CONCLUSIONS: This study highlights the need of males suffering from non-obstructive infertility to have laboratory genetic testing performed. An abnormal finding can have significant consequences to assisted reproductive techniques and fertility treatment, and provide a firm diagnosis to couples with longstanding infertility.
Assuntos
Cromossomos Humanos Y/genética , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/genética , Espermatozoides/citologia , Cariótipo Anormal , Adolescente , Adulto , Azoospermia/genética , Deleção Cromossômica , Análise Citogenética/métodos , Humanos , Síndrome de Klinefelter/diagnóstico , Masculino , Oligospermia/diagnóstico , Estudos RetrospectivosRESUMO
Pharmacogenetic testing is increasingly available from clinical and research laboratories. However, only a limited number of quality control and other reference materials are currently available for many of the variants that are tested. The Association for Molecular Pathology Pharmacogenetic Work Group has published a series of papers recommending alleles for inclusion in clinical testing. Several of the alleles were not considered for tier 1 because of a lack of reference materials. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Material (GeT-RM) program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 18 DNA samples derived from Coriell cell lines. DNA samples were distributed to five volunteer testing laboratories for genotyping using three commercially available and laboratory developed tests. Several tier 2 variants, including CYP2C9∗13, CYP2C19∗35, the CYP2C cluster variant (rs12777823), two variants in VKORC1 (rs61742245 and rs72547529) related to warfarin resistance, and two variants in GGCX (rs12714145 and rs11676382) related to clotting factor activation, were identified among these samples. These publicly available materials complement the pharmacogenetic reference materials previously characterized by the GeT-RM program and will support the quality assurance and quality control programs of clinical laboratories that perform pharmacogenetic testing.
Assuntos
Carboxiliases/genética , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C9/genética , Sistema Enzimático do Citocromo P-450/genética , Farmacogenética , Variantes Farmacogenômicos , Vitamina K Epóxido Redutases/genética , Alelos , Genótipo , Técnicas de Genotipagem , Humanos , Farmacogenética/métodos , Testes FarmacogenômicosRESUMO
Extra copies of the Prader-Willi-Angelman syndrome critical region (PWASCR) have been shown to have detrimental phenotypic effects depending on the parent of origin. Hexasomy for the PWASCR is rare; only 6 cases have been described to date. We report on a 15-year-old girl referred for developmental delay and seizures with a mosaic tricentric small marker chromosome (SMC) 15 identified by routine G-banding chromosome studies. C-banding and FISH confirmed the presence of three chromosome 15 centromeres as well as four copies of the PWASCR on the SMC in approximately 60% of interphase cells. Microsatellite genotyping documented maternal inheritance of the SMC, and methylation-sensitive multiplex ligation-dependent PCR amplification (MS-MLPA) showed that the extra copies of the PWASCR contained on the marker chromosome bear a methylation pattern similar to a normal maternal chromosome, implying maternal inheritance. These findings are consistent with the patient's phenotype as paternal inheritance of such a marker chromosome is thought to be benign. However, this patient's phenotype is the mildest described to date and may be a result of mosaicism for the SMC.
Assuntos
Síndrome de Angelman/genética , Aberrações Cromossômicas , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 15/genética , Impressão Genômica/genética , Síndrome de Prader-Willi/genética , Adolescente , Síndrome de Angelman/patologia , Metilação de DNA , Feminino , Humanos , Hibridização in Situ Fluorescente , Mosaicismo , Mães , Fenótipo , Síndrome de Prader-Willi/patologiaRESUMO
CONTEXT: Deficient anterior pituitary with variable immune deficiency (DAVID) syndrome is a recently described, rare disorder characterized by anterior pituitary hormone deficiencies and common variable immunodeficiency associated with NFKB2 mutations. Posterior pituitary hormone deficiencies have not been reported in patients with DAVID syndrome. CASE DESCRIPTION: We report a pediatric patient who initially presented with hypogammaglobulinemia and alopecia totalis, who was identified to have a de novo NFKB2 mutation at one year of age. He developed central diabetes insipidus and central adrenal insufficiency at three and four years of age, respectively. At seven years of age, he had not developed GH or TSH deficiencies. Whole exome sequencing ruled out known genetic causes of central diabetes insipidus, adrenal insufficiency, and hypopituitarism. CONCLUSION: This is a report of central diabetes insipidus in a patient with DAVID syndrome caused by an NFKB2 mutation. This case report expands the evolving endocrine phenotype associated with NFKB2 mutations beyond anterior pituitary deficiencies.
RESUMO
BACKGROUND: Loss of expression of imprinted genes in the 15q11.2-q13 region is known to cause either Prader-Willi syndrome (PWS) or Angelman syndrome (AS), depending on the parent of origin. In some patients (1 % in PWS and 2-4 % in AS), the disease is due to aberrant imprinting or gene silencing, or both. RESULTS: We report here a 4-year-old boy on whom a chromosomal microarray (CMA) was performed due to mild hand tremors, mild developmental delays, and clumsiness. CMA revealed absence of heterozygosity (AOH) spanning the entire chromosome 15, suggesting uniparental isodisomy 15. The patient had no definitive phenotypic features of PWS or AS. Methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) was performed to determine the parent of origin of the uniparental disomy (UPD) by examining methylation status at maternally imprinted sites. Interestingly, our patient had a mosaic methylation pattern. We identified nine additional previously tested patients with a similar mosaic methylation pattern. CMA was performed on these individuals retrospectively to test whether patients with mosaic methylation are more likely to have UPD of chromosome 15. Of the nine patients, only one had regions of AOH on chromosome 15; however, this patient had numerous regions of AOH on multiple chromosomes suggestive of consanguinity. CONCLUSION: The patients with mosaic methylation had milder or atypical features of AS, and the majority also had some features characteristic of PWS. We suggest that quantitative methylation analysis be performed for cases of atypical PWS or AS. It is also important to follow up with methylation testing when whole-chromosome isodisomy is detected.
RESUMO
Molecular pathology is an essential element of pathology training. As more molecular tests have become available, there is an increasing need for pathology trainees to receive a strong foundation in molecular pathology. Appointed by the Training and Education Committee of the Association for Molecular Pathology, the Molecular Curriculum Task Force has developed a suggested curriculum in molecular pathology for residents. The foundations of molecular pathology are presented as a series of goals and objectives that residency programs can use to develop their educational programs. As pathologists continue to expand their roles to include regular clinical consultations in the realm of molecular testing, a strong foundation in molecular pathology and genomic medicine has become essential to the practice of pathology.
Assuntos
Currículo , Patologia Molecular/educação , Técnicas de Laboratório Clínico , Educação de Pós-Graduação em Medicina , Genômica , Humanos , Infecções , Internato e Residência , Neoplasias , FarmacogenéticaRESUMO
OBJECTIVE: To assess the efficiency of target-enrichment next-generation sequencing (NGS) with copy number assessment in inherited neuropathy diagnosis. METHODS: A 197 polyneuropathy gene panel was designed to assess for mutations in 93 patients with inherited or idiopathic neuropathy without known genetic cause. We applied our novel copy number variation algorithm on NGS data, and validated the identified copy number mutations using CytoScan (Affymetrix). Cost and efficacy of this targeted NGS approach was compared to earlier evaluations. RESULTS: Average coverage depth was â¼760× (median = 600, 99.4% > 100×). Among 93 patients, 18 mutations were identified in 17 cases (18%), including 3 copy number mutations: 2 PMP22 duplications and 1 MPZ duplication. The 2 patients with PMP22 duplication presented with bulbar and respiratory involvement and had absent extremity nerve conductions, leading to axonal diagnosis. Average onset age of these 17 patients was 25 years (2-61 years), vs 45 years for those without genetic discovery. Among those with onset age less than 40 years, the diagnostic yield of targeted NGS approach is high (27%) and cost savings is significant (â¼20%). However, the cost savings for patients with late onset age and without family history is not demonstrated. CONCLUSIONS: Incorporating copy number analysis in target-enrichment NGS approach improved the efficiency of mutation discovery for chronic, inherited, progressive length-dependent polyneuropathy diagnosis. The new technology is facilitating a simplified genetic diagnostic algorithm utilizing targeted NGS, clinical phenotypes, age at onset, and family history to improve diagnosis efficiency. Our findings prompt a need for updating the current practice parameters and payer guidelines.
Assuntos
Variações do Número de Cópias de DNA , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Polineuropatias/genética , Adolescente , Adulto , Idoso , Algoritmos , Criança , Pré-Escolar , Testes Genéticos/economia , Custos de Cuidados de Saúde , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Pessoa de Meia-Idade , Proteína P0 da Mielina/genética , Proteínas da Mielina/genética , Polineuropatias/economia , Adulto JovemRESUMO
Human Epstein-Barr virus (EBV) and cytomegalovirus (CMV) can cause serious complications in immunocompromised patients. Rapid diagnosis of EBV and CMV infection is critical in the management of the disease so that anti-viral therapy can be started early. Here we describe the development of real-time PCR assays using TaqMan probes and molecular beacons and compare the performance of both assays with a well-established, validated, gel-based PCR method for the quantification of EBV and CMV in patients' samples. The TaqMan and molecular beacon assays were linear between 10 to 10(7) viral genomes/reaction. Both assays generated calibration curves with strong correlation and low intra-assay and interassay variation. Results of EBV and CMV viral load determination inpatient samples obtained by the gel-based and real-time PCR were very similar. The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during anti-viral therapy in two of six pediatric patients. The data indicate that these TaqMan and molecular beacon approaches are accurate, rapid, and reliable assays for the diagnosis and monitoring of EBV and CMV infections in patients.
Assuntos
Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/virologia , DNA Viral/análise , DNA Viral/sangue , DNA Viral/genética , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/virologia , Humanos , Técnicas de Sonda Molecular , Sondas de Oligonucleotídeos/genéticaRESUMO
BACKGROUND: Graft-versus-host disease (GVHD) is an important, underdiagnosed cause of mortality associated with liver transplantation. We identified 12 cases of GVHD among 1,082 liver transplantations performed in patients at our institution between 1991 and 1998. Patients typically developed fever, skin rash, diarrhea, or pancytopenia within 2 to 6 weeks after their transplant. Treatment generally involved increased immune suppression and hematopoietic cytokines (granulocyte colony stimulating factor, granulocyte monocyte colony stimulating factor); however, all but one patient died, most often from sepsis. Early in its course, GVHD was difficult to distinguish from cytomegalovirus disease or drug reactions. The diagnosis was confirmed by demonstration of substantial donor lymphoid chimerism. METHODS: To identify risk factors for severe GVHD, a retrospective analysis was performed comparing index cases with the rest of the cases in our institutional experience. RESULTS: Closely matched human leukocyte antigen recipients, those older than 65 years, and recipients with donors more than 40 years younger were at higher risk for GVHD. One case occurred in a patient with a congenital immunodeficiency. CONCLUSIONS: Liver transplant-associated GVHD is a progressive and fatal disease. Future approaches should focus on prevention and might include avoidance of closely matched human leukocyte antigen donors, treatment of the donor to reduce the number of lymphocytes, or reduction of immunosuppression in the early posttransplant period.
Assuntos
Doença Enxerto-Hospedeiro/etiologia , Transplante de Fígado/efeitos adversos , Adolescente , Adulto , Fatores Etários , Idoso , Criança , Pré-Escolar , Feminino , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/terapia , Teste de Histocompatibilidade , Humanos , Síndromes de Imunodeficiência/congênito , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de RiscoRESUMO
The α-thalassemias are a group of hereditary disorders caused by reduced synthesis of the α-chain of hemoglobin. We have developed and tested an α-thalassemia assay that uses both multiplex ligation-dependent probe amplification (MLPA) with Luminex-based detection and deletion PCR technologies. The MLPA assay consisted of 20 probes, 15 of which hybridized to the α-globin gene cluster and 5 that served as control probes. A PCR assay was developed to confirm the presence of heterozygous/homozygous 3.7-kb and 4.2-kb deletions. MLPA and PCR results were compared to Southern blot (SB) results from 758 and 133 specimens, respectively. Lastly, MLPA and PCR results were reviewed and summarized from 5386 clinically tested specimens. SB and MLPA results were concordant in 678/687 (99%) specimens. PCR detected all deletions detected by SB with no false positives. No deletions or duplications were identified in 2630 (49%) clinically tested specimens. Extra α-globin copies were identified in 76 patients. A deletion of one or two α-globin genes was identified in 1251 (23%) and 1349 (25%) specimens, respectively, including 15 different genotypes. A deletion of three (hemoglobin H) and four α-globin genes (Hb Bart's) was observed in 65 or 3 specimens, respectively. Six patients had a deletion within the α-globin regulatory region MCS-R2. Thus, MLPA plus deletion PCR identify multiple α-globin gene deletions/duplications in patients being tested for α-thalassemia.
Assuntos
Sondas de DNA/metabolismo , Deleção de Genes , Família Multigênica/genética , Reação em Cadeia da Polimerase/métodos , alfa-Globinas/genética , Southern Blotting , HumanosRESUMO
OBJECTIVE: To demonstrate a role for microsatellite analysis in the evaluation of prenatal cases with possible placental mesenchymal dysplasia (PMD). METHODS: We present a case report in which several molecular analyses of amniocytes and products of conception were used in combination with cytogenetic and ultrasound studies to evaluate a pregnancy with a clinical suspicion of PMD. A combination of Southern blotting analysis, methylation-sensitive polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) was utilized to evaluate methylation patterns in the Beckwith-Wiedemann syndrome (BWS) and Prader-Willi/Angelman syndrome (PW/AS) critical regions. A series of microsatellite markers were used to evaluate the possibility of an androgenetic cell line. RESULTS: Methylation studies performed for the BWS and PW/AS critical regions were abnormal and consistent with a molecular diagnosis of BWS and Angelman syndrome. Further studies of amniocytes using microsatellite markers identified androgenetic and biparental cell lines in approximately a 10:1 ratio, respectively. CONCLUSIONS: Prenatal ultrasonography, karyotyping and molecular genetic evaluation for BWS alone were not sufficient to identify the underlying etiology of PMD in this case. The androgenetic cell line was only identified after microsatellite analysis.