Early and transient gene expression changes in pressure overload-induced cardiac hypertrophy in mice.

Cardiac hypertrophy is an important risk factor for cardiac morbidity and mortality. To unravel the underlying pathogenic genetic pathways, we hybridized left ventricular RNA from Transverse Aortic Constriction mice at 48 h, 1 week, and 2, 3, and 8 weeks after surgery to microarrays containing a 15K fetal cDNA collection. Key processes involved an early restriction in the expression of metabolic genes, accompanied by increased expression of genes related to growth and reactivation of fetal genes. Most of these genes returned to basal expression levels during the later, compensated hypertrophic phase. Our findings suggest that compensated hypertrophy in these mice is established by rapid adaptation of the heart at the cost of gene expression associated with metabolic activity, with only temporary expression of possible maladaptive processes. Therefore, the transient early changes may reflect a beneficial response to pressure overload, as deterioration of cardiac hemodynamic function or heart failure does not occur.

Sequence-based typing of HLA-B: the B7 cross-reacting group.

The large number of polymorphic sites in the HLA-B locus makes sequencing an efficient way of detecting and analysing them. Most polymorphic sites are located in the alpha1 and alpha2 domains of the molecule, encoded by exons 2 and 3 of the gene. An HLA-B-specific sequence-based typing (SBT) strategy was designed for routine application identifying the polymorphic sites in these domains. Exons 2 and 3 were amplified separately using amplification primers located in intron 1, intron 2 and intron 3. Separate amplification of exons 2 and 3 resulted in short polymerase chain reacting (PCR) products and enabled a solid-phase sequencing approach, which made correct assignment of heterozygous positions possible due to low background. A one-step sequencing reaction was performed using fluorescent dye-labelled sequencing primers. One forward sequencing reaction was performed for exon 2, whereas for exon 3, two forward sequencing reactions were needed using two different sequencing primers located in intron 2 and exon 3. The combined sequences of exon 2 and 3 were used for automatic alignment to an HLA-B sequence database and automatic allele assignment. A total of 355 individuals with at least one allele belonging to the B7 cross-reacting group (B7, 13, 22, 27, 40, 41, 42, 47, 48, 81 and 82) were typed for HLA-B by SBT. In the B7 group 48 different alleles were identified, in the non-B7 group a further 59 alleles were sequenced, 9 new alleles were identified. The sequencing strategy described has proven to be reliable and efficient for high-resolution HLA-B typing.

Strong association between HLA-Cw*0706 and HLA-B*44032 in the Bubi population from Equatorial Guinea.

Unrelated Bubi, native to the island of Bioko (Equatorial Guinea), were previously typed by low-resolution polymerase chain reaction using sequence-specific primers (PCR-SSP) and serology for HLA-A, -B and -C. HLA-B*44 was found frequently and associated with Cw*07. We have studied the HLA subtypes of 20 B*44pos/Cw*07pos Bubi individuals. HLA-B and -C were typed by sequencing exons 2 and 3. To distinguish the alleles Cw*1701/02/03, Cw*07011/012/06 and Cw*1801/02 additional sequencing of exon 1 or 5 was performed. All 20 B*44pos/Cw*07pos individuals of the Bubi population were typed Cw*0706 positive. Nineteen of them carried the B*44032 allele and one B*4407. In addition, 19 B*44neg/ Cw*07pos Bubi individuals were typed for HLA-C and none of them proved Cw*0706 positive. To determine whether the association between Cw*0706 and B*44032 was limited to the Bubi, 19 individuals from Dutch Caucasian families were typed in which B44 and Cw7 segregated on one haplotype. None of these individuals showed the presence of B*44032 or Cw*0706. The haplotypes found in the Dutch Caucasians were B*4402-Cw*0704, B*44031-Cw*07011 and B*44031-Cw*0702. The present observation indicates a strong association between B*44032 and Cw*0706 in the Bubi population.

There is more to HLA-C than exons 2 and 3: sequencing exons 1, 4 and 5.

HLA-C was shown to be a highly polymorphic gene which can be accurately typed for by sequencing methodologies. Most HLA-C sequence-based typing protocols described so far are based on analysis of sequence data of exons 2 and 3. Nonetheless, exons 1, 4 and 5 also contain nucleotide substitutions which contribute to the polymorphisms of the HLA-C locus. Ten alleles contain polymorphic positions in exons 1, 4 and 5, Cw*0701/06, Cw*1202112, Cw*15051/2, Cw*1701/02, and Cw*1801/02. Here we describe a reliable solid-phase sequence-based typing strategy for sequencing exons 1, 4 and 5, which is an extended protocol of our previous HLA-C study. A panel of 16 individuals, carrying 27 different Cw-alleles, was typed for exons 1, 4 and 5 to check the newly designed primers. No allelic dropout or preferential amplification was noticed in these individuals. The panel was also sequenced in order to check the known polymorphisms present in exons 1, 4 and 5. For exon 5 the sequences of the alleles Cw*0302, *0501 and *07011 did not correspond with the published data. In addition, exons 1, 4 and 5 were sequence-based typing typed in 28, 17 and 59 individuals, respectively. Two new alleles were detected which contain polymorphic positions outside exons 2 and 3, Cw*07012 and Cw*1703. The unknown sequence data of exons 1, 4 and 5 of the alleles Cw*02024, *0308, *1506 and *16041 were elucidated. The described high-resolution sequence-based typing protocol for sequencing exons 1, 4 and 5 will be a valuable tool to study the HLA-C locus for polymorphisms outside exons 2 and 3 and for identification of the presently known HLA-C alleles with polymorphic positions in these exons.

Double-target fluorescence in situ hybridization distinguishes multiple genetically aberrant clones in head and neck squamous cell carcinoma.

Genomic heterogeneity has been observed in several solid tumor types. To investigate this phenomenon in head and neck squamous cell carcinoma (HNSCC), we analyzed macroscopically distinct tissue samples of 12 resected tumors by a combination of fluorescence in situ hybridization (FISH) and DNA flow cytometry. Using a panel of centromeric DNA probes, numerical chromosomal aberrations were detected in 10 tumors, 9 of which showed a single DNA aneuploid peak. Imbalances in chromosomal copy numbers resulted in unique patterns of chromosomal aberrations for each tumor case. Two types of tumors could be distinguished, i.e., tumors (n = 5) containing a single aneusomic clone and tumors (n = 5) with multiple aneusomic clones. The center of this latter group of tumors was shown to be genetically more heterogeneous than the tumor margin. In conclusion, this study showed that 1) the pattern of chromosomal aberrations varies greatly between different HNSCC, 2) a major clone with a specific pattern of chromosomal aberrations has spread throughout most HNSCC, and 3) a subgroup of HNSCCs contains additional clones with a different pattern of chromosomal aberrations. Based on these results, HNSCC can be divided into a genetically more homogeneous and a genetically more heterogeneous group.

A reliable and efficient high resolution typing method for HLA-C using sequence-based typing.

Serological typing of HLA-C has been poor and almost half of its alleles are serologically undetectable blanks in most populations. Therefore, DNA typing techniques have been used to identify and type for the HLA-C gene. Sequence-based typing (SBT) has proven a major typing strategy for highly polymorphic HLA genes. The technique enables direct identification of all sequence motifs without the need to continuously adjust primers. Here we describe a reliable solid-phase SBT strategy for HLA-C which can be used to distinguish all currently known HLA-C alleles without prior knowledge gained by low resolution typing. Exons 2 and 3 were amplified and sequenced and if necessary sequences of exons 1 and 5 were determined. A total of 257 individuals were typed for HLA-C using this protocol and 30 of the 42 known HLA-C alleles were detected. All heterozygous combinations found in this study were unambiguously discriminated. One hundred and forty-four individuals from the Dutch population were typed randomly. In this group Cw*0701 and *0702 were the most frequently detected alleles. Of the serological Cw blank alleles Cw*1203 was found to have the highest frequency (16%). From the total group 212 individuals were typed serologically and 106 were retyped with 97 selected antisera to further compare serological and molecular defined phenotypes. Discrepancies between serological typing and SBT are mainly attributable to the serologically Cw blank alleles Cw*12-18. The high resolution SBT protocol described will be a valuable tool for the identification of HLA-C alleles and the determination of the role of HLA-C in marrow and organ transplantation.