Molecular genetic and cell physiological characterisation of an autosomal recessive disorder with a chromosome condensation defect

Titelblatt und Inhaltsverzeichnis Einleitung Material und Methoden Ergebnisse Diskussion Zusammenfassung Literatur AnhangIn der vorliegenden Arbeit wurde der Nachweis geführt, dass die erste beim Menschen beschriebene Chromosomenkondensationsstörung auf Mutationen im MCPH1-Gen beruht. Die Patienten sind klinisch durch ausgeprägte Mikrozephalie und mentale Retardierung charakterisiert. Proliferierende Gewebe der Patienten weisen einen zellulären Phänotyp mit einem erhöhten Anteil Prophase-ähnlicher Zellen (PLCs) von bis zu 20% auf. Dieser Defekt beruht auf einer vorzeitigen Chromosomenkondensation in der G2 Phase des Zellzyklus und auf einer verzögerten postmitotischen Dekondensation in der G1 Phase. Das MCPH1-Gen kodiert für das Protein Microcephalin. Dieses enthält drei BRCT (BRCA1 C-terminus)-Domänen und ein nukleares Lokalisationssignal. Durch RNA Interferenz gegen Microcephalin-mRNA in humanen Kontrollzellen konnte der beschriebene zelluläre Phänotyp imitiert und somit gezeigt werden, dass der Funktionsverlust von Microcephalin auch ursächlich für die Chromosomenkondensationsstörung ist. Die Analysen des Verhaltens von Zellkulturen der Patienten nach ionisierender Bestrahlung zeigten, dass es bei diesen nicht zum Verlust des DNA-Schadenskontrollpunktes kommt, sondern der Zellzyklus am G2/M-Übergang vor dem Eintritt in die Mitose arretiert wird. Die physiologisch normale Reaktion nach DNA-Schädigung, nämlich eine Akkumulation von Zellen am G2/M-Übergang, spiegelt sich bei den Patientenzellen auch in einer Erhöhung des Anteils Prophase-ähnlicher Zellen nach Bestrahlung wieder. Dieser Effekt kann zur Differenzialdiagnose bei Patienten mit primärer Mikrozephalie eingesetzt werden. Die Beschreibung des zellulären Phänotyps und die Aufklärung des Basisdefektes ermöglichten auch die Diagnosestellung bei einem Patienten mit sehr mildem klinischen und zellulären Phänotyp. Dieser Patient weist eine missense-Mutation (T27R) in der ersten BRCT-Domäne von Microcephalin auf. Die klinische Symptomatik dieses Patienten entspricht nicht den Einschlusskriterien für die klinische Diagnose von primärer Mikrozephalie (MCPH). Daher erfordert dieser Befund eine Überarbeitung der Definition der klinischen Symptomatik für MCPH. Der Chromosomenkondensationsdefekt wird durch eine Fehlregulation des Condensin II Komplexes vermittelt und ist unabhängig von Condensin I. Es wurde nachgewiesen, dass der Anteil PLCs durch RNA Interferenz gegen Untereinheiten des Condensin II Komplexes - jedoch nicht gegen Condensin I-spezifische Untereinheiten - in Patientenzellen deutlich reduziert wird. Da dies, wie mit Zellzyklusphasen-spezifischen Markern gezeigt wurde, sowohl aus einer Abnahme von Zellen mit kondensiertem Chromatin in der G2-Phase als auch der G1-Phase des Zellzyklus resultiert, wurde damit außerdem zum ersten Mal eine Funktion von Condensin II in der Chromosomendekondensation demonstriert. Die Ergebnisse der RNAi-Experimente sind im Einklang mit den Resultaten aus Immunfluoreszenzanalysen mit Antikörpern gegen die Condensinkomplexe. Condensin II ist im Gegensatz zu Condensin I zum Zeitpunkt der fehlregulierten Chromosomenkondensation nuklear und bindet vorzeitig an die zentrale Chromatidachse.In the presented dissertation it was demonstrated that the first chromosome condensation disorder described in human is caused by mutations in the MCPH1 gene. Patients are characterised by pronounced microcephaly and mental retardation. Proliferating tissues of the patients display a cellular phenotype with an enhanced fraction of prophase-like cells (PLCs) of up to 20 %. The defect is due to premature chromosome condensation in the G2 phase of the cell cycle and due to a delayed decondensation in G1 phase. MCPH1 encodes microcephalin, a protein containing three BRCT (BRCA1 C-terminus)-domains and a nuclear localisation signal. The specific cellular phenotype can be reproduced by RNA interference against microcephalin-mRNA in human control cells demonstrating that the functional loss of microcephalin is the underlying defect causing the misregulation of chromosome condensation. Analyses of the cell cycle behaviour of cell lines of the patients after ionising irradiation revealed that DNA damage checkpoint control is not disturbed, but rather the cell cycle is arrested at the G2/M transition preceding mitosis. The observed normal physiological reaction of accumulation of cells at G2/M transition following DNA damage is reflected in the patient cells by a distinct increase of the proportion of prophase-like cells - an effect that can be used for differential diagnosis of patients with primary microcephaly. The description of the cellular phenotype in combination with the definition of the underlying mutation lead to the diagnosis of a patient with an extraordinarily mild clinical and cellular phenotype. This patient has a missense mutation (T27R) in the first BRCT domain of microcephalin. The clinical symptoms of this patient do not correspond to the criteria hitherto used for diagnosis of autosomal recessive primary microcephaly (MCPH). Therefore, these findings demand a revision of the definition of the symptomatic complex of MCPH. The condensation defect is mediated by misregulation of the condensin II complex but is independent of condensin I. It could be demonstrated that the fraction of prophase-like cells can be markedly reduced by RNA interference against condensin II subunits, but not by silencing of condensin I specific subunits. As shown using cell cycle phase specific markers, this is due to a decrease of cells displaying condensed chromatin in both G2- and G1-phase of the cell cycle, demonstrating for the first time a function of condensin II in the decondensation process. The results of the RNAi experiments are in accordance with the findings of immunofluorescence analyses with antibodies against the condensin complexes. Unlike condensin I, condensin II stays in the nucleus of the prophase-like cells and binds prematurely to the central chromatid axis

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

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

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

Cell cycle-dependent regulation of MCPH1 transcripts.

<p>(A) HeLa cells were arrested in G1 phase by double thymidine block. Cultures harvested at various time points after release were analyzed using flow cytometry. (B) Plots represent numbers of cells as a function of their DNA content. A total of 90% of the cells synchronously progressed into S phase (0–4 h), entered G2 phase (4–6 h), started passing through mitosis after 7 h, and were completely in G1 phase after 12 h. (C) Levels of MCPH1-FL (diamonds, solid line), MCPH1Δe9–14 (squares, dotted line), and MCPH1Δe8 (circles, dashed line) mRNA. Data represent means ± S.E.M. of three independent experiments and are normalized to the expression levels of <i>GAPDH</i> and <i>B2M</i>.</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

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

Inter-laboratory study on standardized MPS libraries : evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA

We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer’s recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework