A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.Peer reviewe

A Novel MCPH1 Isoform Complements the Defective Chromosome Condensation of Human MCPH1-Deficient Cells

Biallelic mutations in MCPH1 cause primary microcephaly (MCPH) with the cellular phenotype of defective chromosome condensation. MCPH1 encodes a multifunctional protein that notably is involved in brain development, regulation of chromosome condensation, and DNA damage response. In the present studies, we detected that MCPH1 encodes several distinct transcripts, including two major forms: full-length MCPH1 (MCPH1-FL) and a second transcript lacking the six 39 exons (MCPH1De9–14). Both variants show comparable tissue-specific expression patterns, demonstrate nuclear localization that is mediated independently via separate NLS motifs, and are more abundant in certain fetal than adult organs. In addition, the expression of either isoform complements the chromosome condensation defect found in genetically MCPH1-deficient or MCPH1 siRNA-depleted cells, demonstrating a redundancy of both MCPH1 isoforms for the regulation of chromosome condensation. Strikingly however, both transcripts are regulated antagonistically during cell-cycle progression and there are functional differences between the isoforms with regard to the DNA damage response; MCPH1-FL localizes to phosphorylated H2AX repair foci following ionizing irradiation, while MCPH1De9–14 was evenly distributed in the nucleus. In summary, our results demonstrate here that MCPH1 encodes different isoforms that are differentially regulated at the transcript level and have different functions at the protein level

Y-chromosomal analysis identifies the skeletal remains of Swiss national hero Jörg Jenatsch (1596–1639)

Abstract not availableCordula Haas, Natallia Shved, Frank Jakobus Rühli, Christina Papageorgopoulou, Josephine Purps, Maria Geppert, Sascha Willuweit, Lutz Roewer, Michael Krawcza

Complementation of PCC in patient fibroblasts.

<p>Cells are derived from patient with homozygous truncating mutation c.427dupA (p.T143NfsX5) in <i>MCPH1</i>. Chromosome preparations from (A) non-transduced cells and (B) cells expressing GFP only, or GFP fusions with (C) full-length, (D) Δe9–14, (E) Δe8, or (F) Δe1–7 MCPH1. Arrows indicate nuclei of prophase-like cells (PLCs). (G) Mean rates of PLCs (filled columns) of slides from A-F. Open columns represent mean mitotic indices. Error bars denote the S.D. of counts of approximately 1000 cells each from three independent experiments.</p

Intracellular distribution of MCPH1 isoforms.

<p>(A) MCPH1-deficient fibroblasts expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fractionated and cytoplasmic (Cyt) and nuclear (Nuc) protein extracts were analyzed using immunoblotting with an antibody against GFP. The nuclear matrix protein p84 and GAPDH were used as index proteins and loading controls. (B) Cells indicated in A stained with an anti-GFP antibody (green), counterstained with DAPI (blue) and analyzed using fluorescence microscopy. Arrows indicate the prophase-like nuclei. Scale bar = 10 µm. All MCPH1 isoforms exhibit unambiguous nuclear localization.</p

Colocalization of MCPH1 and γH2AX in ionizing irradiation-induced nuclear foci.

<p>(A) Non-transduced (NT) MCPH1-deficient 562T cells and 562T stably expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fixed 2 h after irradiation with 10 Gy and co-stained with antibodies against γH2AX (red) and GFP (green). Nuclei were counterstained with DAPI (blue). Rectangles frame areas, which are shown enlarged in the bottom row. MCPH1 focus formation was observed for MCPH1 isoforms containing the C-terminal BRCT tandem. (B) Quantification of cells expressing foci containing γH2AX and/or (C) MCPH1. Error bars indicate the S.D. of three different measurements, counting approximately 300 nuclei. * <i>p</i>≤0.05 vs. NT as calculated using the Student's <i>t</i>-test.</p

The human <i>MCPH1</i> gene, its transcripts and predicted polypeptides.

<p>(A) Exon (filled boxes) and intron (open boxes) organization of the 241 906-bp encompassing <i>MCPH1</i> gene locus. Red arrows indicate the positions of the regular and of the alternative (*) polyadenylation sites (polyA). (B) The full-length (FL) and the alternative transcripts Δe9–14, Δe1–3, and Δe8: numbered boxes indicate exons, black filled areas illustrate the entire coding regions (CDS), and colored areas show untranslated regions (UTR) as indicated. (C) Predicted polypeptides representing MCPH1 isoforms: blue boxes depict the positions of BRCT domains, while green boxes represent the site of the canonical nuclear localization signal sequence (NLS). Two additional amino acids, S and M, are included into MCPH1Δe9–14 prior to premature termination (#). (D) Expression of MCPH1 transcript variants. Columns represent the levels of MCPH1 transcripts in indicated adult and fetal tissues determined using quantitative real-time PCR. Data represent means ± one S.D. of three independent experiments and are normalized to the geometric mean levels of <i>UBC</i>, <i>GAPDH</i>, <i>B2M</i>, and <i>HPRT1</i> cDNA.</p

Expression of GFP-tagged MCPH1 isoforms in MCPH1-deficient 562T fibroblasts.

<p>(A) Cells were transduced with GFP-tagged coding sequence of full-length MCPH1 cDNA in a conditional, doxycycline (DOX)-dependent construct with a second regulatory construct trKRAB. Cultures were exposed to increasing DOX concentrations as indicated. Whole-cell extracts were prepared 72 h later and analyzed for the expression of MCPH1 using immunoblotting with an antibody against GFP. (B) The graph shows MCPH1 band intensity relative to the loading control p84 plotted against DOX concentrations. Data represent means ± one S.D. of three independent assays. (C) Immunoblot analysis of whole-cell extracts from non-transduced (NT) 562T cells (lane 1), 562T cells transduced with the regulatory construct only (lane 2), with GFP alone (30 kDa, lane 3) or with GFP fused to MCPH1-FL (120 kDa, lane 4), MCPH1Δe9–14 (94 kDa, lane 5), MCPH1Δe8 (78 kDa, lane 6), or MCPH1Δe1–7 (96 kDa, lane 7) with an antibody against GFP.. Nuclear matrix protein p84 served as the loading control.</p

Nuclear localization signals (NLSs) in human MCPH1.

<p>(A) The positions of the putative NLS motifs and their amino acid sequences are highlighted in the diagram of the full-length MCPH1. (B) Subcellular distribution of GFP-tagged wild-type (wt) MCPH1 and mutants with deleted NLSs as indicated were transiently expressed in HeLa cells. Cytoplasmic (Cyt) and nuclear (Nuc) protein extracts were immunoblotted with an antibody against GFP (left panel). GAPDH (center panel) and the nuclear matrix protein p84 (right panel) served as index proteins and loading controls. (C) Ratios of relative GFP band intensity in the cytoplasmic (Cyt) vs. nuclear (Nuc) fractions. Absolute numbers were assessed using densitometry and normalized to the loading controls. Columns designate means, and error bars represent the S.D. from three different experiments. Significant differences to wt MCPH1 are indicated by asterisks denoting <i>p</i><0.05 (Student's <i>t</i>-test). Scale bar = 10 µm.</p