Expanding the clinical and molecular spectrum of ATP6V1A related metabolic cutis laxa.

Several inborn errors of metabolism show cutis laxa as a highly recognizable feature. One group of these metabolic cutis laxa conditions is autosomal recessive cutis laxa type 2 caused by defects in v-ATPase components or the mitochondrial proline cycle. Besides cutis laxa, muscular hypotonia and cardiac abnormalities are hallmarks of autosomal recessive cutis laxa type 2D (ARCL2D) due to pathogenic variants in ATP6V1A encoding subunit A of the v-ATPase. Here, we report on three affected individuals from two families with ARCL2D in whom we performed whole exome and Sanger sequencing. We performed functional studies in fibroblasts from one individual, summarized all known probands' clinical, molecular, and biochemical features and compared them, also to other metabolic forms of cutis laxa. We identified novel missense and the first nonsense variant strongly affecting ATP6V1A expression. All six ARCL2D affected individuals show equally severe cutis laxa and dysmorphism at birth. While for one no information was available, two died in infancy and three are now adolescents with mild or absent intellectual disability. Muscular weakness, ptosis, contractures, and elevated muscle enzymes indicated a persistent myopathy. In cellular studies, a fragmented Golgi compartment, a delayed Brefeldin A-induced retrograde transport and glycosylation abnormalities were present in fibroblasts from two individuals. This is the second and confirmatory report on pathogenic variants in ATP6V1A as the cause of this extremely rare condition and the first to describe a nonsense allele. Our data highlight the tremendous clinical variability of ATP6V1A related phenotypes even within the same family

Loss of murine Gfi1 causes neutropenia and induces osteoporosis depending on the pathogen load and systemic inflammation.

Gfi1 is a key molecule in hematopoietic lineage development and mutations in GFI1 cause severe congenital neutropenia (SCN). Neutropenia is associated with low bone mass, but the underlying mechanisms are poorly characterized. Using Gfi1 knock-out mice (Gfi1-ko/ko) as SCN model, we studied the relationship between neutropenia and bone mass upon different pathogen load conditions. Our analysis reveals that Gfi1-ko/ko mice kept under strict specific pathogen free (SPF) conditions demonstrate normal bone mass and survival. However, Gfi1-ko/ko mice with early (nonSPF) or late (SPF+nonSPF) pathogen exposure develop low bone mass. Gfi1-ko/ko mice demonstrate a striking rise of systemic inflammatory markers according to elevated pathogen exposure and reduced bone mass. Elevated inflammatory cytokines include for instance Il-1b, Il-6, and Tnf-alpha that regulate osteoclast development. We conclude that low bone mass, due to low neutrophil counts, is caused by the degree of systemic inflammation promoting osteoclastogenesis

TFE3 activation in a TSC1‐altered malignant PEComa: challenging the dichotomy of the underlying pathogenic mechanisms

Perivascular epithelioid cell tumors (PEComas) form a family of rare mesenchymal neoplasms that typically display myomelanocytic differentiation. Upregulation of mTOR signaling due the inactivation of TSC1/2 (Tuberous Sclerosis 1 and 2) is believed to be a key oncogenic driver in this disease. Recently, a subgroup of PEComas harboring TFE3 (Transcription Factor E3) rearrangements and presenting with a distinctive morphology has been identified. TSC1/2 and TFE3 aberrations are deemed to be mutually exclusive in PEComa, with two different pathogenic mechanisms assumed to lead to tumorigenesis. Here, we challenge this dichotomy by presenting a case of a clinically aggressive TCS1-mutated PEComa displaying a TFE3-altered phenotype. FISH analysis was suggestive of a TFE3 inversion; however, RNA and whole genome sequencing was ultimately unable to identify a fusion involving the gene. However, a copy number increase of the chromosomal region encompassing TFE3 was detected and transcriptome analysis confirmed upregulation of TFE3, which was also seen at the protein level. Therefore, we believe that the TSC1/2-mTOR pathway and TFE3 overexpression can simultaneously contribute to tumorigenesis in PEComa. Our comprehensive genetic analyses add to the understanding of the complex pathogenic mechanisms underlying PEComa and harbor insights for clinical treatment options

Gfi1 regulates hematopoietic and mesenchymal cells. Low bone mass in Gfi1-ko/ko mice results from inflammatory response due to severe neutropenia.

<p>Loss of Gfi1 and housing at conditions of variable pathogen load lead to increased mortality, inflammatory response, and reduction of bone mass. The inflammatory response is the main determinant of osteopenia and osteoporosis in the severe neutropenia mouse model Gfi1.</p

Gfi1-ko/ko mutants upon SPF+nonSPF housing show elevated osteoclast activity and bone cell marker expression.

<p><b>(A)</b> In Gfi1-ko/ko mutant mice kept under SPF and SPF+nonSPF Rankl plasma levels are unaffected and Opg is elevated compared to Gfi1-wt/wt mice. Bone resorption (CTX-I) is mildly increased in Gfi1-ko/ko mice under both conditions. Bone formation (PINP) is mildly reduced in SPF Gfi1-ko/ko mice compared to Gfi1-wt/wt but not under SPF+nonSPF conditions. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01. <b>(B)</b> Relative expression of bone marker mRNA was analyzed with qPCR and Gapdh was used as endogenous control. Results are presented as ratio Gfi1-ko/ko vs. Gfi1-wt/wt (n = 3 with 3 technical replicates/sample; error bar indicates SD). Normal expression is indicated with the dotted line at 1. All Ob. specific markers such as Col1a1, Runx2, and Spp1 are approx. 2-fold elevated. <b>(C)</b> Markers indicating mature Oc. such as Acp5 and Ctsk are approx. 4-fold increased. Interestingly, the marker for Oc. progenitor cells Sfip1 (Pu.1) is also 4-fold elevated. Relative expression of Oc. marker mRNA was analyzed with qPCR and Gapdh was used as endogenous control.</p

Elevated G-CSF and GM-SCF but normal M-CSF levels in Gfi1-ko/ko mice.

<p>The plasma levels of M-CSF, G-CSF, and GM-CSF were assessed by ELISA in mice kept at nonSPF, SPF, and SPF+nonSPF conditions. <b>(A)</b> M-CSF levels of Gfi1-ko/ko mutants were unaffected compared to controls upon all breeding conditions. <b>(B)</b> G-CSF is massively elevated in Gfi1-ko/ko mice compared to controls housed at nonSPF, SPF, and SPF+nonSPF conditions. <b>(C)</b> At all conditions Gfi1-ko/ko mice demonstrate significantly elevated GM-CSF levels. GM-CSF levels were measured by Q-Plex ELISA assay. The GM-CSF was below the limit of detection (b.l.d.) in Gfi1-wt/wt under all breeding conditions. Error bars represent SD. <b>(D)</b> The plasma levels of inflammatory cytokines were assessed by Q-Plex assay in control and Gfi1-ko/ko mice kept at nonSPF, SPF, and SPF+nonSPF conditions. Inflammatory cytokines such as Il-1β, Il-6, IFN-gamma, and TNF-alpha are significantly elevated in Gfi1-ko/ko mice kept at SPF+nonSPF conditions compared to their controls. Highest levels of Il-1β, Il-6, IFN-gamma, and TNF-alpha are present in Gfi1-ko/ko mice exclusively kept at nonSPF conditions. Due to sample limitations we measured for nonSPF mice serum pools (n = 2 with n = 4/pool) that were not statistically evaluated. Please note that lowest cytokine concentrations in Gfi1-ko/ko mice are present upon SPF conditions. In control mice IFN-gamma was below the limit of detection independent from the breeding condition. Full data set of analyzed cytokines is available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s011" target="_blank">S5 Table</a>. Error bars represent SEM. Statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p

Diminished osteoblast and osteoclast activity in nonSPF Gfi1-ko/ko mice.

<p><b>(A)</b> The Ob. and Oc. activity in Gfi1-ko/ko and Gfi1-wt/wt mice kept at nonSPF conditions was determined by measuring the plasma markers receptor activator of NF-kB ligand (Rankl), osteoprogerin (Opg), cross-linked carboxy-terminal telopeptide of type I collagen (CTX-I), and osteocalcin. Elevated Opg and diminished Rankl levels in Gfi1-ko/ko mutants suggest reduced osteoclast activity. Diminished bone resorption and formation in Gfi1-ko/ko mice is indicated by lowered CTX-I and osteocalcin levels, respectively. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01. <b>(B)</b> Basal expression of Gfi1 mRNA in different tissues shows highest values in spleen, bone marrow, and cortical bone. Non-haematopoietic tissues such as liver or cartilage and <i>in vitro</i> differentiated Oc. as well as Ob. demonstrated very low Gfi1 mRNA expression (n = 3 with 3 technical replicates/sample). Gfi1 expression was assessed with quantitative PCR (qPCR) and Gapdh was used as endogenous control. <b>(C)</b> All markers of Ob. proliferation and differentiation are diminished in Gfi1-ko/ko mutant lysates. Please note the low levels of osteoblast markers Ocn, Runx2, and Osx but normal expression of the osteocyte marker Dmp1. Values are shown as ratio of Gfi1-ko/ko vs. Gfi1-wt/wt mRNA expression (n = 3 with 3 technical replicates/sample). Normal expression is indicated with the dotted line at 1. Relative expression of bone marker genes was assessed by qPCR in full RNA preparations of cortical bone. Gapdh was used as endogenous control. <b>(D)</b> Immunohistology of the Ob. marker osteocalcin (Bglap) demonstrates abundant signals (red) in controls at the bone marrow to bone junction but low levels in Gfi1-ko/ko mutants. Please note abundant osteocalcin signals also in bone marrow cells. Immunostaining was performed on film supported cryosections. Counterstaining occurred with DAPI visualizing cell nuclei.</p

Differential bone marrow cell count of Gfi1 mice kept under SPF and SPF+nonSPF conditions.

<p>Differential bone marrow cell count of Gfi1 mice kept under SPF and SPF+nonSPF conditions.</p

Gfi1-ko/ko mice kept under nonSPF conditions develop osteopenia.

<p><b>(A)</b> Trabecular bone of vertebrae was measured with microCT. Gfi1-ko/ko mice kept under nonSPF conditions developed severe osteopenia as indicated by 60% BV/TV reduction compared to Gfi1-wt/wt mice. Upon SPF breeding Gfi1-ko/ko mice show a 15% reduction in BV/TV. However, SPF+nonSPF conditions induced intermediate osteopenia characterized by approx. 30% BV/TV reduction. Please note Gfi1-ko/ko mice kept at nonSPF conditions develop cortical bone osteopenia in femur. <b>(B)</b> Representative bone sections stained with von Kossa/ Kernechtrot illustrate trabecular bone in vertebrae of mice kept under nonSPF, SPF and SPF+nonSPF conditions. <b>(C)</b> Quantification of bone tissue by histomorphometry revealed reduced BV/TV values in Gfi1-ko/ko vertebrae under nonSPF and SPF+nonSPF conditions compared to controls. Breeding within the SPF environment did not significantly affect bone mass of Gfi1-ko/ko mice. <b>(D)</b> Histomorphometric quantification of the osteoblast covered bone surface (Ob.S/BS) shows reduced but elevated values upon nonSPF and SPF+nonSPF breeding, respectively. SPF breeding did not affect Ob.S/BS between control and mutant mice. <b>(E)</b> Quantification of the osteoclast covered bone surface (Oc.S/BS) revealed diminished but raised values in Gfi1-ko/ko mice upon nonSPF and SPF+nonSPF breeding compared to their corresponding Gfi1-wt/wt controls, respectively. Gfi1-ko/ko mutants grown under SPF conditions showed elevated Oc.S/BS counts compared to Gfi1-wt/wt mice. For better comparability, Gfi1-wt/wt values were set to 1 and Gfi1-ko/ko values were relatively calculated for each breeding condition. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s008" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s009" target="_blank">S3</a> Tables for absolute values. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p

Housing conditions determine Gfi1-ko/ko mice body mass and survival.

<p><b>(A)</b> Average growth curves from control mice kept under nonSPF conditions indicate evolving development also beyond weaning (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3). However, soon after weaning (P19) Gfi1-ko/ko mutants display significantly delayed growth. Development under SPF growth conditions did not affect growth of Gfi1-ko/ko mice (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3). Upon SPF+nonSPF housing Gfi1-ko/ko mice demonstrated relative normal growth compared to controls (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3); however, Gfi1-ko/ko mice show significant body mass reduction. All curves show values of male mice. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s007" target="_blank">S1 Table</a> for health monitoring. <b>(B)</b> At SPF and SPF+nonSPF conditions Gfi1-ko/ko mice showed a mortality rate of approx. 9% and 6%, respectively. <b>(C)</b> Final body mass of controls and Gfi1-ko/ko mice was assessed at indicated time points for SPF and SPF+nonSPF conditions. Combined SPF+nonSPF breeding caused a body mass reduction of approx. 25% in male and female Gfi1-ko/ko mice. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p