Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Congenital heart block maternal sera autoantibodies target an extracellular epitope on the α1G T-type calcium channel in human fetal hearts.

Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB.We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene) in the AV junction of human fetal hearts compared to the apex (18-22.6 weeks gestation). Using human fetal hearts (20-22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305-319 of the extracellular loop linking transmembrane segments S5-S6 in α1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells.Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets

Affinity-purified maternal CHB serum antibodies co-localize with α_1G.

<p>(<b>A</b>) Cardiomyocytes from ventricle of human fetal hearts, gestational week 20.6, were dissociated, immunofluorescence stained, and visualized with confocal microscopy. Cardiomyocytes are stained for Cav3.1 (α_1G) (Red), for the cardiomyocyte marker anti-Troponin-T (Green), and nuclei visualized with DAPI (Blue). Secondary antibodies alone gave no stain. Inset image demonstrates expression on the surface in a cross section image. Images shown are representative of various cardiomyocyte samples; similar staining patterns were obtained in four replicate experiments. Secondary antibodies, anti-rabbit-IgG-Cy3 (Red) and anti-mouse-IgG-Alexa488 (green) alone give no stain (data not shown). Note that DAPI stains the nuclei of cardiomyocytes and of other cells present in the samples, such as fibroblasts. Scale bar represents 12 µm. (<b>B</b>) Dissociated and non-permeabilized cardiomyocytes from ventricle of 20.6 week human fetal hearts, immunofluoresence stained for α_1G affinity-purified serum (red), and α_1G (green). Co-localization of staining (yellow) was assessed with confocal microscopy. Scale bar represents 12 µm.</p

T-type calcium channel α_1G is expressed in the AV node of human fetal hearts.

<p>(<b>A</b>) Masson's trichrome staining of 21-week human fetal heart demonstrates morphology of the AVJ region with light pink staining of the spindle-like cells in the AV node (AVN) and the AV bundle (AVB), green staining corresponding to collagen fibres. (<b>B</b>) α_1G staining (Red) is present in AVJ and AV bundle regions which are distinguishable by positive NF-160 staining (Green). Scale bars represent 100 µm (A) and 150 µm (B).</p

Maternal sera antibody profile screening of α_1G peptides.

<p>Maternal sera from one CHB⁺ pregnancy (<b>A, C, E</b>) and one CHB⁻ pregnancy (<b>B, D, F</b>) were tested in ELISA against 15aa long overlapping peptides derived from extracellular regions of α_1G: T-type aa130–380 (<b>A, B</b>); aa774–963 (<b>C, D</b>); and, aa1308–1536 (<b>E, F</b>). Reactivity above threshold (HC: Average+3×St. Dev; set at 0.59, 0.64, 0.48 respectively for A/B, C/D, and E/F) was observed in the CHB⁺ but not CHB⁻ serum to α_1G peptides p305 (aa305–319, light grey bar) and p315 (aa315–323, dark grey bar) was observed in the CHB⁺ but not the CHB⁻ serum. Above threshold reactivity was also observed among peptides in the region spanning aa1308–1536.</p

Expression analysis, and CHB maternal sera immune reactivity towards α_1G.

<p>(<b>A</b>) Although both α_1G and α_1H are expressed in human fetal hearts, real time PCR demonstrates that transcripts from <i>CACNA1G</i> (α_1G) are 2.2- to 4-fold higher in the AVJ than in the apex tissue (between 18–22.6 weeks gestation), whereas <i>CACNA1H</i> expression levels are between 0.7- and 1.1-fold in the AVJ compared to the apex. (<b>B</b>) Combining the data from 3 hearts (18.0 weeks-22.6 weeks), shows that <i>CACNA1G</i> expression in the AVJ is significantly higher than <i>CACNA1H</i> in the AVJ (p<0.05). (<b>C</b>) Western blot with human fetal heart lysate (20.4 weeks) demonstrates α_1G expression by a Cav3.1 commercial antibody (lane 1), that is blocked by the peptide immunogen of this antibody (aa1–22 of rat α_1G) (lane 2). CHB sera (anti-Ro and anti-La titer >100 IU) also binds to α_1G (lane 3); this reactivity is blocked by the α_1G p305 peptide (lane 4). Sera from mothers with anti-Ro/La antibodies (anti-Ro = 60 IU and anti-La titer >100 IU) giving birth to normal babies do not have immune reactivity to the α_1G protein (lane 5), and a commercial α_1H antibody confirms that the band seen is not α_1H. (<b>D</b>) α_1G isolated from human fetal heart (AV junction, ventricle, and apex) was immunoprecipitated with a Cav3.1 antibody, and the immunoblot was probed with human sera. Immunoblot was subsequently stripped and re-probed with a second Cav3.1 antibody towards a different epitope, demonstrating presence and specificity of the IP towards α_1G. Note that CHB sera is defined as anti-Ro^/La positive sera from pregnancies affected by CHB. Normal sera are from pregnancies with a healthy outcome and not affected by CHB.</p

Reactivity in CHB pregnancies is specific for the p305 peptide of α_1G and can be blocked.

<p>Four overlapping 15aa peptides, and one 20aa peptide were selected to further characterize maternal sera reactivity to α_1G in mothers with CHB pregnancies (CHB⁺) compared to mothers with unaffected pregnancies (CHB⁻). Reactivity to (<b>A</b>) peptide p300 (aa300–314), (<b>B</b>) p305 (aa305–319), (<b>C</b>) p310 (aa310–324), (<b>D</b>) p315 (aa315–329) demonstrate that sera from mothers with pregnancies affected by CHB (CHB⁺), have significantly higher p305 antibody levels compared to unaffected (CHB⁻) pregnancies (p<0.05). Although p305 (aa305–319) had the highest reactivity, a longer peptide combining p305 and p310 designated p305/310 (aa305–24) demonstrated a significant difference between CHB⁺ and CHB⁻ maternal serum (<b>E</b>). Pre-incubation of one CHB⁺ maternal sera, and one healthy control (HC) sera with increasing concentration (0, 20, 40 µg/ml) of peptide p305 (aa305–319) demonstrates that the reactivity of sera is specific for this peptide and can be blocked (<b>F</b>). Error bars indicate mean ± SE.</p

Sequence identity and maternal sera reactivity for α_1G and α_1H T-type calcium channel peptides.

<p>(<b>A</b>) Alignment of peptides derived from human α_1G and α_1H S5–S6 extracellular loop I sequences. The ‘core sequence’ of interest with 10 amino acids in common among the α_1G peptides is underlined, and the seven residues that are identical in the α_1G and α_1H sequences are indicated in bold type. Note that this group is present in the sequence of each immunoreactive peptide, whereas the non-reactive peptides lack two or more of these amino acid residues. Sequences shown correspond to human CAC1G (α_1G) and human CAC1H (α_1H) (UniProt accession numbers O43497 and 095180, respectively). (<b>B</b>) Peptides derived from the aligned loop regions of α_1G and α_1H exhibit a similar pattern of CHB⁺/CHB⁻ reactivity. CHB maternal sera ELISA reactivity (OD 405 nm) was plotted with interleaved bars for comparison. Error bars represent Mean ± SE. (<b>C</b>) Reactivity of CHB⁺ maternal sera was observed to be significantly higher than HC sera, where p<0.05 for both α_1G p305 (aa305–319) and α_1H p330 (aa330–343), but did not reach significance for comparison with CHB⁻ sera (p = 0.075 and p = 0.184 for α_1G and α_1H, respectively).</p

Ion channel blocker effects on Wenckebach cycle length in atrial paced newborn and adult rabbit hearts.

<p>Wenckebach cycle length (WBCL) was recorded during perfusion with calcium channel and <i>I</i>_f channel blockers in newborn and adult rabbit hearts (WBCL in milliseconds). Equivalent effects were observed in adult and newborn hearts treated with L-type calcium channel blockers (<b>A</b>) Diltiazem and (<b>B</b>) Verapamil and with the <i>I</i>_f blocker ZD7288 (<b>C</b>) (p = <i>ns</i> repeated measures ANOVA). The T-type calcium channel blocker Mibefradil (<b>D</b>) demonstrated significant preferential AV block in the newborn hearts as compared to the adult hearts (p<0.05). Values shown are mean ± SEM, newborn (n = 7) and adult (n = 8). Diltiazem showed a trend toward a difference in sensitivity of adult and newborn hearts, however there was great variability in results between experiments. (<b>E</b>) Effects of the α_1H T-type calcium channel blocker NiCl₂ were also investigated on WBCL in newborn Langendorff rabbit hearts. WBCL measurements do not differ from each other significantly with mean ± SEM (repeated measures ANOVA p = <i>ns</i>). One heart out of 6 that appeared to be an outlier showed some variability in response to NiCl₂, and was excluded from statistical analysis.</p