Journal: Human Molecular Genetics

Article Title: POMK mutations disrupt muscle development leading to a spectrum of neuromuscular presentations

doi: 10.1093/hmg/ddu296

Figure Lengend Snippet: Severe compound heterozygous changes in POMK cause WWS. (A) Family 2 was an Italian trio including a boy affected by WWS (P3). (B) One of the diagnostic features of WWS, cobblestone lissencephaly with hydrocephalus, was noted upon MRI imaging. (C) Exome sequencing identified two heterozygous mutations in POMK, which were confirmed by Sanger sequencing. One change was a 1 bp deletion leading to a frameshift (c.286delT) and the second change was a missense affecting a highly conserved amino acid (c.905T > A) (D). (E) The truncating mutations in Families 1 and 2 are found very early in the protein and are both predicted to completely remove the putative kinase domain in POMK.

Article Snippet: SGK196 expression analysis cDNA samples from human fetal and adult postmortem brain tissue were obtained commercially (BioChain and Clontech). qPCR was performed using SYBR green reagents (SsoFast EvaGreen Supermix from Bio-Rad) on a Bio-Rad platform (CFX384) using the following primers SGK196-For 5′-CTTCTTCATCGCTCCTCGACA-3′ and SGK196-Rev 5′-AGCCAAGGTGAGCAGTTTTTC-3′.

Techniques: Diagnostic Assay, Imaging, Sequencing

Journal: Human Molecular Genetics

Article Title: POMK mutations disrupt muscle development leading to a spectrum of neuromuscular presentations

doi: 10.1093/hmg/ddu296

Figure Lengend Snippet: POMK is highly expressed during muscle and brain development. (A and B) Quantitative PCR analysis of POMK expression in human tissues shows highest expression in brain, skeletal muscle, kidney and heart in fetal and adult tissues. Highest expression in the brain is observed in the cerebral cortex. (C) Cortical expression was studied in the mouse and found to be dynamic increasing after E16.5. (D) The expression analysis of POMK in the developing human muscle was also revealed to be dynamic. In fetal muscle cells, POMK is found in both laminin-positive (i) and laminin-negative cells (arrow in ii). Scale bar: 10 µm (E) Lamin A/C staining outlines the nuclear envelope and POMK decorates perinuclear and cytoplasmic structures in all cells in the fetal muscle. Scale bar: 50 µm (F) In the adult human muscle, POMK expression is restricted to interstitial cells between the myofibers and in cells closely associated with the blood vessels (yellow arrowheads). Scale bar: 50 µm.

Article Snippet: SGK196 expression analysis cDNA samples from human fetal and adult postmortem brain tissue were obtained commercially (BioChain and Clontech). qPCR was performed using SYBR green reagents (SsoFast EvaGreen Supermix from Bio-Rad) on a Bio-Rad platform (CFX384) using the following primers SGK196-For 5′-CTTCTTCATCGCTCCTCGACA-3′ and SGK196-Rev 5′-AGCCAAGGTGAGCAGTTTTTC-3′.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining

Journal: Human Molecular Genetics

Article Title: POMK mutations disrupt muscle development leading to a spectrum of neuromuscular presentations

doi: 10.1093/hmg/ddu296

Figure Lengend Snippet: POMK knockdown reduces dystroglycan glycosylation but does not affect myotube formation. (A) POMK is highly expressed in both MCAM-positive and MCAM-negative cells (arrow) purified from human fetal muscle. MCAM-positive cells are myogenic cells and we wondered whether POMK knockdown would affect the early stages of myocyte development. Scale bars: 10 µm. (B) Purified myocytes will fuse to form myotubes (arrows) over the first week in vitro. Scale bar: 100 µm. (C) POMK is highly expressed starting at Day 1 after myogenic cell purification. (D) A highly efficient siRNA pool was tested and shown to generate consistent knockdown starting at a concentration of 25 nm. (E) No effect was observed in myocyte fusion during the first week in vitro, but α-DG glycosylation was reduced following POMK knockdown. Day 4 samples are shown.

Article Snippet: SGK196 expression analysis cDNA samples from human fetal and adult postmortem brain tissue were obtained commercially (BioChain and Clontech). qPCR was performed using SYBR green reagents (SsoFast EvaGreen Supermix from Bio-Rad) on a Bio-Rad platform (CFX384) using the following primers SGK196-For 5′-CTTCTTCATCGCTCCTCGACA-3′ and SGK196-Rev 5′-AGCCAAGGTGAGCAGTTTTTC-3′.

Techniques: Knockdown, Glycoproteomics, Purification, In Vitro, Concentration Assay

Journal: Human Molecular Genetics

Article Title: POMK mutations disrupt muscle development leading to a spectrum of neuromuscular presentations

doi: 10.1093/hmg/ddu296

Figure Lengend Snippet: pomk knockdown disrupts muscle development in the zebrafish embryo. (A) pomk MO injection affects brain, eye and muscle development in the zebrafish embryo. The tail is thickened and the muscle appears disorganized, the head is smaller and retinal fusion is delayed (compare distance between small arrows in WT and MO right panels). Scale bar: 500 µm. (B) Locomotor activity in the developing zebrafish embryo is disrupted at 20 hpf. Embryo body movement is tracked in a video (Supplementary Material, videos): head movement is tracked in blue and tip of the tail movement in yellow. Both head and tail change position dramatically in the wild type due to spontaneous tail coiling. In the pomk morphants, the head and tail always remain in the same relative situation because the embryo is unable to shift position within the chorion. (B) Morphant larvae at 3 dpf show a shorter and bent tail. Scale bar: 500 µm. (C) pomk morphants muscle at 3 dpf shows signs of muscular dystrophy, laminin and dystrophin (dystr) staining are reduced and glycosylated α-DG is almost completely missing. Disorganization of the muscle fibers can also be noted by the disruption in nuclear orientation revealed by Hoechst staining. Scale bar: 100 µm. (E) Injection of human full-length POMK mRNA in the pomk morphant embryo causes significant phenotypic improvement, while injection of truncated (Q109*) POMK mRNA does not (left graph). Overexpression of mRNA up to 1 ng has no effect on the embryos (right graph). Results are from at least three independent experiment and are quantified at 1 dpf scoring the phenotypes shown in Figure 5A. Embryos scored as severe are greatly underdeveloped with barely distinguishable head and tail.

Article Snippet: SGK196 expression analysis cDNA samples from human fetal and adult postmortem brain tissue were obtained commercially (BioChain and Clontech). qPCR was performed using SYBR green reagents (SsoFast EvaGreen Supermix from Bio-Rad) on a Bio-Rad platform (CFX384) using the following primers SGK196-For 5′-CTTCTTCATCGCTCCTCGACA-3′ and SGK196-Rev 5′-AGCCAAGGTGAGCAGTTTTTC-3′.

Techniques: Knockdown, Injection, Activity Assay, Staining, Disruption, Over Expression

Journal: bioRxiv

Article Title: Evolution of sex hormone-binding globulin gene expression in the primate testis

doi: 10.1101/2024.08.05.606716

Figure Lengend Snippet: RT-PCR assays for SHBG transcripts (exons 1A-8 and 1-8) were performed on cDNA derived from HepG2 cells, rhesus macaque, lion-tailed macaque and baboon testes. Analyses for TNP1 and Vimentin transcripts indicate the presence of germ and Sertoli cells, respectively, in testis-derived cDNA. The integrity of all cDNA samples is assessed by analysis for GAPDH transcripts. All PCR products were subjected to electrophoresis on either 1% (for SHBG ) or 1.5% (for TNP1 , Vimentin and GAPDH ) agarose gels. Molecular sizes in bp are indicated for the SHBG assays.

Article Snippet: Non-human primate testis cDNA samples were a generous gift from Dr. Rüdiger Behr at the Deutsches Primatenzentrum in Göttingen, Germany.

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Electrophoresis

Journal: bioRxiv

Article Title: Evolution of sex hormone-binding globulin gene expression in the primate testis

doi: 10.1101/2024.08.05.606716

Figure Lengend Snippet: RT-PCR assays for SHBG transcripts (exons 3-8) were performed on cDNA derived from HepG2 cells, and three marmoset testes. Analyses for TNP1 and Vimentin transcripts indicate the presence of germ and Sertoli cells, respectively, in testis-derived cDNA. The integrity of all cDNA samples is assessed by analysis for GAPDH transcripts. All PCR products were subjected to electrophoresis on either 1% (for SHBG ) or 1.5% (for TNP1 , Vimentin and GAPDH ) agarose gels. Molecular sizes in bp are indicated for the SHBG assay.

Article Snippet: Non-human primate testis cDNA samples were a generous gift from Dr. Rüdiger Behr at the Deutsches Primatenzentrum in Göttingen, Germany.

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Electrophoresis

Journal: bioRxiv

Article Title: Evolution of sex hormone-binding globulin gene expression in the primate testis

doi: 10.1101/2024.08.05.606716

Figure Lengend Snippet: RT-PCR assays for SHBG transcripts (exons 3-8) were performed on cDNA derived from liver and testes from two grey mouse lemurs ( Microcebus murinus ). Analyses for TNP1 and Vimentin transcripts indicate the presence of germ and Sertoli cells, respectively, in testis-derived cDNA. The integrity of all cDNA samples is assessed by analysis for GAPDH transcripts. All PCR products were subjected to electrophoresis on either 1% (for SHBG ) or 1.5% (for TNP1 , Vimentin and GAPDH ) agarose gels. Molecular sizes in bp are indicated for the SHBG assay. RT, reverse transcriptase.

Article Snippet: Non-human primate testis cDNA samples were a generous gift from Dr. Rüdiger Behr at the Deutsches Primatenzentrum in Göttingen, Germany.

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Electrophoresis, Reverse Transcription