RESUMEN
BACKGROUND AND OBJECTIVES: Several comprehensive genotyping platforms for determining red blood cell (RBC) antigens have been established and validated for use in the Caucasian and Black populations, but not for the Chinese. The multiplex ligation-dependent probe amplification (MLPA) assay was validated for RHD genotyping in the Chinese. MATERIALS AND METHODS: The blood samples of 200 D+, 200 D- and 62 D variant Chinese donors were collected. RhD antigen was routinely typed by serological method. D variant phenotype was determined by an anti-D panel (D-Screen), when RBCs were available. The RHD genotype and its zygosity were analysed with the RH-MLPA technique. When the MLPA was unable to identify a RHD variant, direct sequencing of all exons of the RHD gene was performed. RESULTS: In 200 D+ donors, DD (168/200, 84%), D (12/200, 6%), DDD genotype (1/200) and D variant allele carriers (19/200, 9·5%) were found. In 200 D- donors, six reported RHD alleles, RHD*01EL.01, RHD*01N.03, RHD*01N.05, RHD*01N.16, RHD*DFR2 and RHD*weak partial 15 and one novel RHD*1154T allele were identified in 36·5% (73/200) of them. In 62 D variant donors, three novel RHD alleles, RHD*79_81delCTC, RHD*710T and RHD*689A, and twelve reported alleles, RHD*DVI.3, RHD*weak partial 15, RHD*DVI.4, RHD*01EL.01, RHD*01N.03, RHD*DLO, RHD*DV.5, RHD*D-CE(2-10), RHD*730C, RHD*weak D type 25, 33 and 72, were identified, either alone or in combination. CONCLUSION: The RH-MLPA assay correctly identified the common RHD variant alleles in the Chinese population. However, DNA sequencing was required to identify certain alleles; probes to detect these alleles should be added into the assay.
Asunto(s)
Donantes de Sangre , Genotipo , Técnicas de Diagnóstico Molecular/normas , Reacción en Cadena de la Polimerasa Multiplex/normas , Sistema del Grupo Sanguíneo Rh-Hr/genética , Pueblo Asiatico/genética , Exones , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , FenotipoRESUMEN
OBJECTIVES: We aim to elucidate causes of false-positive fetal RHD screening results obtained with cell-free DNA. METHODS: Fetal RHD screening was performed in 32,222 samples from RhD-negative women by multiplex real-time PCR in triplicate for RHD exons 5 and 7 using cell-free DNA isolated from maternal plasma obtained in the 27th gestational week. PCR results were compared with cord blood serology in 25,789 pregnancies (80.04%). False-positive cases were analyzed. Known biological causes (RHD variant genes), technical causes of discordance, and errors around blood sampling were investigated with leukocyte DNA from maternal and cord blood, and cell-free DNA from stored maternal plasma. RESULTS: Not only RHD but also Y-chromosome (DYS14) sequences were present in four plasma samples from RHD-negative women bearing an RHD-negative girl. Sample mix-up and other sampling errors could be excluded in three samples. CONCLUSIONS: These results indicate that false-positive fetal RHD screening results can be caused by cell-free DNA fragments in maternal plasma derived from a third cell line that is not representative for either the maternal genome or the genome of the vital fetus. We propose that remaining (cyto)trophoblasts of a vanishing twin are the underlying mechanism, and we estimate a frequency of this phenomenon of 0.6%.
Asunto(s)
Incompatibilidad de Grupos Sanguíneos/diagnóstico , Pruebas de Detección del Suero Materno , Embarazo Gemelar/sangre , Sistema del Grupo Sanguíneo Rh-Hr/sangre , Biomarcadores/sangre , Incompatibilidad de Grupos Sanguíneos/sangre , Incompatibilidad de Grupos Sanguíneos/genética , Incompatibilidad de Grupos Sanguíneos/inmunología , Reacciones Falso Positivas , Femenino , Sangre Fetal , Técnicas de Genotipaje , Humanos , Recién Nacido , Masculino , Reacción en Cadena de la Polimerasa Multiplex , Embarazo , Embarazo Gemelar/genética , Embarazo Gemelar/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Sistema del Grupo Sanguíneo Rh-Hr/genéticaRESUMEN
Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.
Asunto(s)
Donantes de Sangre , Antígenos de Grupos Sanguíneos/genética , Tipificación y Pruebas Cruzadas Sanguíneas/métodos , Tamizaje Masivo/métodos , Antígenos de Plaqueta Humana/análisis , Antígenos de Plaqueta Humana/genética , Antígenos de Grupos Sanguíneos/análisis , Sistemas de Computación , Genotipo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Polimorfismo Conformacional Retorcido-Simple , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We have recently cloned a novel Doublecortin CaMK-like kinase (rDCAMKL) cDNA, and a related cDNA called CaMK-related peptide (CARP) from the rat hippocampus. These genes are structurally highly similar to the human DCAMKL-1 gene and doublecortin, a gene associated with X-linked lissencephaly and subcortical band heterotopia. Here we report on the genomic organization of the murine DCAMKL gene and its products. Our results show that DCAMKL and CARP are alternative splice products of the same gene. The DCAMKL gene also generates three alternatively-spliced rDCAMKL transcripts of which we have cloned the corresponding cDNAs and which potentially generate different DCAMKL proteins. In situ hybridization experiments show that the different rDCAMKL transcripts are all expressed in the adult rat hippocampus. We conclude that alternative splicing of the DCAMKL gene may generate different but similar proteins in the adult rat hippocampus thereby regulating different but overlapping aspects of DCAMKL controlled neuronal plasticity.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Hipocampo/fisiología , Proteínas Asociadas a Microtúbulos , Transcripción Genética/fisiología , Factores de Edad , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Proteínas Portadoras , Citoesqueleto/enzimología , ADN Complementario/aislamiento & purificación , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Exones , Femenino , Expresión Génica/fisiología , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Neuropéptidos/genética , Fosfoproteínas/genética , RatasRESUMEN
BACKGROUND: Since the cloning of the gene for autosomal dominant polycystic kidney disease type 2 (PKD2), approximately 40 different mutations of that gene have been reported to be associated with the disease. The relationship between the PKD2 genotype and phenotype, however, remains unclear. METHODS: Detailed clinical information was collected for PKD2 families in which the underlying mutation had been identified. Logistic regression analysis was employed to assess the influence of age and sex on hypertension, hematuria, renal calculi, and urinary tract infections, and a clinical phenotype score was computed. Patients were then grouped according to the relative location of their mutation within the cDNA sequence, and differences in the mean phenotypic score between groups were tested for statistical significance by means of a multiple pairwise t-test. RESULTS: While phenotypic scores for each mutational group revealed a considerable degree of intragroup variability, the variability in phenotypic scores was significantly higher between mutational groups than within groups. A group-wise comparison of the mean phenotypic scores confirmed the observation of significant nonlinear variation in disease severity, with high- and low-scoring mutational groups interspersed along the gene sequence. CONCLUSION: The identification of groups of mutations in the PKD2 gene, which differ significantly with respect to clinical outcome, is to our knowledge the first description of a genotype/phenotype correlation in autosomal dominant polycystic kidney disease. It also provides evidence against complete loss of function of the mutant PKD2 gene product.
Asunto(s)
Mapeo Cromosómico , Proteínas de la Membrana/genética , Mutación/genética , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/fisiopatología , Análisis de Varianza , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Masculino , Dinámicas no Lineales , Fenotipo , Análisis de Regresión , Índice de Severidad de la Enfermedad , Canales Catiónicos TRPPRESUMEN
Autosomal Dominant Polycystic Kidney Disease (ADPKD), a common inherited disease leading to progressive renal failure, can be caused by a mutation in either the PKD1 or PKD2 gene. Both genes encode for putative transmembrane proteins, polycystin-1 and polycystin-2, which show significant homology to each other and are believed to interact at their carboxy termini. To identify genes that code for related proteins we searched for homologous sequences in several databases and identified one partial cDNA and two genomic sequences with significant homology to both polycystin-1 and - 2. Further analysis revealed one novel gene, PKD2L2, located on chromosome band 5q31, and two recently described genes, PKD2L and PKDREJ, located on chromosome bands 10q31 and 22q13.3, respectively. PKD2L2 and PKD2L, which encode proteins of 613 and 805 amino acids, are approximately 65% similar to polycystin-2. The third gene, PKDREJ, encodes a putative 2253 amino acid protein and shows about 35% similarity to both polycystin-1 and polycystin-2. For all the genes expression was found in testis. Additional expression of PKD2L was observed in retina, brain, liver and spleen by RT-PCR. Analyses of five ADPKD families without clear linkage to either the PKD1 or PKD2 locus showed no linkage to any of the novel loci, excluding these genes as the cause of ADPKD in these families. Although these genes may not be involved in renal cystic diseases, their striking homology to PKD2 and PKD1 implies similar roles and may contribute to elucidating the function of both polycystin-1 and polycystin-2.
Asunto(s)
Proteínas de Unión al Calcio/genética , Cromosomas Humanos Par 5 , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Mutación , Fosfoproteínas , Riñón Poliquístico Autosómico Dominante/genética , Proteínas/genética , Receptores de Superficie Celular , Secuencia de Aminoácidos , Canales de Calcio , Bandeo Cromosómico , Bases de Datos Factuales , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Programas Informáticos , Canales Catiónicos TRPPRESUMEN
It is estimated that approximately 15% of families with autosomal dominant polycystic kidney disease (ADPKD) have mutations in PKD2. Identification of these mutations is central to identifying functionally important regions of gene and to understanding the mechanisms underlying the pathogenesis of the disorder. The current study describes mutations in six type 2 ADPKD families. Two single base substitution mutations discovered in the ORF in exon 14 constitute the most COOH-terminal pathogenic variants described to date. One of these mutations is a nonsense change and the other encodes an apparent missense variant. Reverse transcription-PCR from patient lymphoblast RNA showed that, in addition, both mutations resulted in out-of-frame splice variants by activating cryptic splice sites via different mechanisms. The apparent missense variant produced such a strong splicing signal that the processed transcript from the mutant chromosome did not contain any of the normally spliced, missense product. A third mutation, a nonconservative missense change effecting a negatively charged residue in the third transmembrane span, is likely pathogenic and defines a highly conserved residue consistent with a potential channel subunit function for polycystin-2. The remaining three mutations included two frame shifts resulting from deletion of one or two bases in exons 6 and 10, respectively, and a nonsense mutation due to a single base substitution in exon 4. The study also defined a novel intragenic polymorphism in exon 1 that will be useful in analyzing "second hits" in PKD2. Finally, the study demonstrates that there are reduced levels of normal polycystin-2 protein in lymphoblast lines from PKD2-affected individuals and that truncated mutant polycystin-2 cannot be detected in patient lymphoblasts, suggesting that the latter may be unstable in at least some tissues. The mutations described will serve as critical reagents for future functional studies in PKD2.
Asunto(s)
Proteínas de la Membrana/genética , Mutación , Riñón Poliquístico Autosómico Dominante/genética , Empalme del ARN , Humanos , Polimorfismo Genético , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Catiónicos TRPPRESUMEN
Recently the second gene for autosomal dominant polycystic kidney disease (ADPKD), located on chromosome 4q21-q22, has been cloned and characterized. The gene encodes an integral membrane protein, polycystin-2, that shows amino acid similarity to the PKD1 gene product and to the family of voltage-activated calcium (and sodium) channels. We have systematically screened the gene for mutations by single-strand conformation-polymorphism analysis in 35 families with the second type of ADPKD and have identified 20 mutations. So far, most mutations found seem to be unique and occur throughout the gene, without any evidence of clustering. In addition to small deletions, insertions, and substitutions leading to premature translation stops, one amino acid substitution and five possible splice-site mutations have been found. These findings suggest that the first step toward cyst formation in PKD2 patients is the loss of one functional copy of polycystin-2.
Asunto(s)
Genes/genética , Proteínas de la Membrana/genética , Mutación/genética , Riñón Poliquístico Autosómico Dominante/genética , Adulto , Membrana Celular/química , Cromosomas Humanos Par 4/genética , Análisis Mutacional de ADN , Exones/genética , Humanos , Proteínas de la Membrana/química , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-Simple , Empalme del ARN , Canales Catiónicos TRPPRESUMEN
A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding domain.
Asunto(s)
Proteínas de la Membrana/genética , Riñón Poliquístico Autosómico Dominante/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Canales de Calcio/química , Canales de Calcio/genética , Mapeo Cromosómico , Cromosomas Humanos Par 4 , Clonación Molecular , Secuencia de Consenso , Cristalografía por Rayos X , Femenino , Glicosilación , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Mutación , Linaje , Fenotipo , Polimorfismo Conformacional Retorcido-Simple , Proteínas/química , Proteínas/genética , Canales de Sodio/química , Canales de Sodio/genética , Canales Catiónicos TRPPRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder A mutation in at least three different genes can cause the disease. A mutation in the first gene, the PKD1 gene, which has been identified on chromosome 16p13.3, accounts for ADPKD in approximately 86% of the families with this disorder. In the majority of the other ADPKD families the disease is caused by a mutation in a second gene, the PKD2 gene. This gene has been mapped to chromosome 4q21-22, but has not yet been identified. In a few families ADPKD is not caused by a mutation in either the PKD1 or the PKD2 gene. The locus for a possible third gene has not yet been determined. Now that haplotype analysis with polymorphic markers at the ADPKD1 and ADPKD2 loci is possible, we can easily distinguish between both forms of ADPKD. We describe a large Dutch family in which ADPKD is linked to chromosome 4. Compared with ADPKD1 families, the disease in this family tends to run a milder course, as has been described previously for other ADPKD2 families.
Asunto(s)
Riñón Poliquístico Autosómico Dominante/clasificación , Riñón Poliquístico Autosómico Dominante/genética , Adulto , Anciano , Anciano de 80 o más Años , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 4 , Femenino , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Linaje , FenotipoRESUMEN
The first three introns of CRK2, a cdc2-homologous gene, have been compared in a total of seven Plasmodium species. The introns were located at conserved sites, suggesting an ancestral origin. Interspecific comparison of intron sequences agreed with the previously inferred evolutionary relationships of the malaria parasites. Unlike the introns in the rodent malaria species, the similarity of the CRK2 introns was regionalized between the human parasite P. vivax and the simian parasite P. knowlesi: the central region of all three introns showed markedly less interspecific similarity than the 5' and 3' regions. This was also in contrast with the organisation and composition of homologous intron pairs from other genes of the same two species. No conservation at the level of secondary structure could be detected, even between highly similar introns. A database search for intron-containing Plasmodium genes was performed. All introns obtained in this way plus the additional CRK2 introns were scanned for the presence of putative branching site consensus sequences. For P. falciparum, we present an update of the 5' and 3' splice-site consensus.