RESUMO
BACKGROUND: Celiac disease (CD) is an inflammatory disorder of the small intestine induced by gluten ingestion. CD has a strong genetic association with human leukocyte antigen (HLA)-DQ2.5 and HLA-DQ8. The absence of HLA-DQ2.5 and HLA-DQ8 has a strong negative predictive value for CD. Genetic screening of HLA-DQ2.5 and HLA-DQ8 in patients at risk is of great value. METHODS: We designed, developed, and validated a multiplex assay based on multiplex ligation-dependent probe amplification (MLPA) technology, allowing the simultaneous detection of DQA1*05-DQB1*02, encoding HLA-DQ2.5, and DQA1*03-DQB1*03:02, encoding HLA-DQ8. The amplified products were separated and identified using capillary electrophoresis. RESULTS: When compared with a polymerase chain reaction followed by single-strand conformation polymorphism/ heteroduplex analysis, one discrepancy was found. Sequencing analysis showed that the developed MLPA assay result was correct. Furthermore, we demonstrated that the MLPA method is able to distinguish between the heterozygote and homozygote expression of HLA-DQ2.5 or HLA-DQ8. CONCLUSIONS: This study shows that it is possible to rapidly and accurately screen for the absence of HLA-DQ2.5 and HLA-DQ8 using MLPA, excluding patients at risk for CD for further serological or histological follow-up. In addition, MLPA might be an accurate tool to screen for other specific HLA types in the context of disease association in a diagnostic laboratory setting.
Assuntos
Doença Celíaca/diagnóstico , Doença Celíaca/imunologia , Antígenos HLA-DQ/análise , Antígenos HLA-DQ/biossíntese , Reação em Cadeia da Polimerase Multiplex/métodos , Doença Celíaca/genética , Eletroforese Capilar/métodos , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Humanos , Reação em Cadeia da Polimerase/métodos , Polimorfismo Conformacional de Fita Simples , Fatores de Risco , Análise de Sequência de DNARESUMO
BACKGROUND: Human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. The B27 allele is present in 90% of patients with this disease, whereas it is present in only 9% of Caucasians. Molecular detection of HLA-B27 is traditionally based on allele specific amplification of exon 2 (Olerup method) or exon 3 (Dominguez method) by PCR, followed by gel analysis. METHODS: We developed a real-time TaqMan PCR based on the Dominguez method with a ß-Globin PCR as internal control. RESULTS: A total of 544 clinical samples were used to compare the real-time TaqMan PCR with the traditional Dominguez PCR, the traditional Olerup PCR and a commercial Olerup based HLA-B27 detection kit (Olerup SSPTM HLA-B27, GenoVision). While 542 samples gave concordant results, two samples showed discrepancies and were further analyzed. One sample that showed a discrepancy was negative with the traditional Olerup method and positive with the three other procedures. Sequencing analysis showed the presence of HLA-B*2712 in this sample. The other sample, positive with both Olerup based PCRs and negative with both Dominguez based methods, turned out to be positive for HLA-B*2707 by sequence analysis. CONCLUSIONS: With a correct result for 543 out of 544 samples (99.8%), we consider our real-time HLA-B27 PCR is a reliable method to detect HLA-B27 in the Dutch population, with reduced hands-on time and contamination risk compared to traditional PCR methods.
Assuntos
Antígeno HLA-B27/genética , Reação em Cadeia da Polimerase em Tempo Real , População Branca/genética , Alelos , Éxons , Predisposição Genética para Doença , Humanos , Países Baixos , Análise de Sequência de DNA , Espondilite Anquilosante/genéticaRESUMO
BACKGROUND: Genotyping of HLA-DQ2.2, HLA-DQ2.5, and HLA-DQ8 is important in celiac disease (CD). The absence of these three genotypes has a strong negative predictive value. METHODS: We designed multiplex ligation-dependent probe amplification (MLPA) for the combined detection of HLA-DQ2.2, HLA-DQ2.5, and HLA-DQ8. The MLPA probe mix was validated against a set of 59 samples characterized by conventional techniques. RESULTS: The MLPA assay genotyped all 59 samples correctly when compared to the results obtained by PCR-SSCP/HD or PCR-SSO and PCR-SSP. CONCLUSION: The MLPA assay provides a reliable single-reaction analysis of the CD risk genotypes HLA-DQ2.2, HLA-DQ2.5, and HLA-DQ8 allowing for stratification or exclusion of disease risk.
Assuntos
Doença Celíaca/genética , Antígenos HLA-DQ/genética , Alelos , Doença Celíaca/sangue , Frequência do Gene/genética , Predisposição Genética para Doença , Genótipo , Antígenos HLA-DQ/sangue , Haplótipos , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Fatores de RiscoRESUMO
BACKGROUND: The success of minimally invasive parathyroidectomy is attributed to evolving preoperative imaging techniques and intraoperative parathyroid hormone (IOPTH) measurement. The additional value of IOPTH measurement in patients undergoing surgery for primary hyperparathyroidism (pHPT) was evaluated. METHODS: Between 1999 and 2010 there were 119 patients who underwent surgery for pHPT at our institutions. In all patients, preoperative imaging was performed and IOPTH samples were collected prospectively but the results were not disclosed during surgery. RESULTS: Postoperative calcium level normalized in 114 patients (96%). The 5 surgical failures represented the maximum yield of IOPTH sampling. Three of these patients would have been identified intraoperatively by an inadequate IOPTH decrease, whereas IOPTH decreased inaccurately in the other 2 patients. In addition, in 1 of these 3 patients no abnormal gland was found during minimally invasive parathyroidectomy and subsequent conventional neck exploration. Therefore, only 2 reoperations would have been prevented (1.7%). CONCLUSIONS: IOPTH would have changed the outcome in 2 patients, increasing the biochemical cure rate from 96% to 98%. We believe that although it can be helpful in certain cases, it may not be necessary routinely in patients treated for pHPT.