Meire Aguena

Rare variants in SOS2 and LZTR1 are associated with Noonan syndrome

Background Noonan syndrome is an autosomal dominant, multisystemic disorder caused by dysregulation of the RAS/mitogen activated protein kinase (MAPK) pathway. Heterozygous, pathogenic variants in 11 known genes account for approximately 80% of cases. The identification of novel genes associated with Noonan syndrome has become increasingly challenging, since they might be responsible for very small fractions of the cases. Methods A cohort of 50 Brazilian probands negative for pathogenic variants in the known genes associated with Noonan syndrome was tested through whole-exome sequencing along with the relatives in the familial cases. Families from the USA and Poland with mutations in the newly identified genes were included subsequently. Results We identified rare, segregating or de novo missense variants in SOS2 and LZTR1 in 4% and 8%, respectively, of the 50 Brazilian probands. SOS2 and LZTR1 variants were also found to segregate in one American and one Polish family. Notably, SOS2 variants were identified in patients with marked ectodermal involvement, similar to patients with SOS1 mutations. Conclusions We identified two novel genes, SOS2 and LZTR1, associated with Noonan syndrome, thereby expanding the molecular spectrum of RASopathies. Mutations in these genes are responsible for approximately 3% of all patients with Noonan syndrome. While SOS2 is a natural candidate, because of its homology with SOS1, the functional role of LZTR1 in the RAS/MAPK pathway is not known, and it could not have been identified without the large pedigrees. Additional functional studies are needed to elucidate the role of LZTR1 in RAS/MAPK signalling and in the pathogenesis of Noonan syndrome.

Transcriptional analysis of the pst operon of Escherichia coli

Abstract. The pst operon of Escherichia coli, which encodes the phosphate-specific transport system, is composed of five genes, pstS, pstC, pstA, pstB and phoU, whose transcription is induced by phosphate starvation. A phosphate-regulated promoter located upstream of the most proximal gene (pstS) controls the transcription of the entire operon. Though the full-length pst mRNA could be detected by an improved RT-PCR protocol, Northern analysis using several pst-specific probes failed to reveal this transcript. Instead, smaller but distinct pst mRNA species were evident. Primer-extension experiments localized the 5′ ends of pst mRNAs within the operon. The data suggest that the full-length mRNA is rapidly processed post-transcriptionally.

Fat grafts supplemented with adipose-derived stromal cells in the rehabilitation of patients with craniofacial microsomia.

Background: Although first reports of the clinical use of adipose-derived stromal cells suggest that this approach may be feasible and effective for soft-tissue augmentation, there is a lack of randomized, controlled clinical trials in the literature. Thus, this study aimed to investigate whether a faster protocol for isolation of adipose-derived stromal cells and their use in combination with fat tissue improve the long-term retention of the grafts in patients with craniofacial microsomia. Methods: Patients with craniofacial microsomia (n = 14) were grafted either with supplementation of adipose-derived stromal cells (experimental group) or without supplementation of adipose-derived stromal cells (control group). The number of viable cells isolated before and after the supplementation of the grafts was calculated, and these cells were examined for mesenchymal cell surface markers using flow cytometry. Computed tomography was performed to assess both hemifaces preoperatively and at 6 months postoperatively. Results: The average number of viable cells isolated before and after the supplementation of the grafts was 5.6 × 105 and 9.9 × 105 cells/ml of fat tissue (p = 0.015). Flow cytometric analysis revealed that the adipose-derived stromal cells were positive for mesenchymal cell markers (>95 percent for CD73 and CD105). Surviving fat volume at 6 months was 88 percent for the experimental group and 54 percent for the control group (p = 0.003). Conclusion: These results suggest that this strategy for isolation and supplementation of adipose-derived stromal cells is effective, safe, and superior to conventional lipoinjection for facial recontouring in patients with craniofacial microsomia. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

Optimization of Parameters for a More Efficient Use of Adipose-Derived Stem Cells in Regenerative Medicine Therapies

Adipose tissue-derived stem cells (ASCs) association to fat in autologous lipotransfer is promising for a more effective soft tissue reconstruction, and optimization of protocols to isolate ASCs from lipoaspirate fat is much needed. We demonstrated that an increase in adipocyte differentiation is dependent on the number of ASCs. In a sample of 10 donors, we found a higher concentration of nucleated cells in the lower abdomen compared to flank (P = 0.015). In a sample of 6 donors we did not find differences in the cell yield obtained by manual or pump-assisted aspiration (P = 0.56). We suggest that the increase in the number of ASCs in the reinjected fat may enhance the efficiency of newly formed adipose tissue and that the anatomical region from which to harvest fat tissue needs to be considered to optimize the number of ASCs in the harvested tissue. Finally, pump-assisted aspiration can be used without any significant harm to the viability of cells.

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

Genetic contribution for non-syndromic cleft lip with or without cleft palate (NS CL/P) in different regions of Brazil and implications for association studies†

Non‐syndromic cleft lip with or without cleft palate (NS CL/P) is a complex disease in which heritability estimates vary widely depending on the population studied. To evaluate the importance of genetic contribution to NS CL/P in the Brazilian population, we conducted a study with 1,042 families from five different locations (Santarém, Fortaleza, Barbalha, Maceió, and Rio de Janeiro). We also evaluated the role of consanguinity and ethnic background. The proportion of familial cases varied significantly across locations, with the highest values found in Santarém (44%) and the lowest in Maceió (23%). Heritability estimates showed a higher genetic contribution to NS CL/P in Barbalha (85%), followed by Santarém (71%), Rio de Janeiro (70%), Fortaleza (64%), and Maceió (45%). Ancestry was not correlated with the occurrence of NS CL/P or with the variability in heritability. Only in Rio de Janeiro was the coefficient of inbreeding significantly larger in NS CL/P families than in the local population. Recurrence risk for the total sample was approximately 1.5–1.6%, varying according to the location studied (0.6–0.7% in Maceió to 2.2–2.8% in Barbalha). Our findings show that the degree of genetic contribution to NS CL/P varies according to the geographic region studied, and this difference cannot be attributed to consanguinity or ancestry. These findings suggest that Barbalha is a promising region for genetic studies. The data presented here will be useful in interpreting results from molecular analyses and show that care must be taken when pooling samples from different populations for association studies.

Craniosynostosis in pycnodysostosis: Broadening the spectrum of the cranial flat bone abnormalities†

Pycnodysostosis is a rare autosomal recessive skeletal dysplasia caused by the absence of active cathepsin K, which is a lysosomal cysteine protease that plays a role in degrading the organic matrix of bones, acting in bone resorption and bone remodeling. The disease is primarily characterized by osteosclerosis, bone fragility, short stature, acro‐osteolysis, and delayed closure of the cranial sutures. A differing feature, cranial synostosis, has occasionally been described in this disorder. We reviewed six unrelated patients with pycnodysostosis (mean age of 10 years and 4 months) in order to evaluate the presence of craniosynostosis. In addition to the typical findings of the condition, they all presented premature fusion of the coronal suture. Although none of them showed signs of cranial hypertension, one patient had had the craniosynostosis surgically corrected previously. These data suggest that the cranial sutures in pycnodysostosis can display contradictory features: wide cranial sutures, which are commonly described, and craniosynostosis. The clinical impact of this latter finding still remains to be elucidated. Further studies are necessary to address more precisely the role of cathepsin K in suture patency.

Transcriptional Processing of the pst Operon of Escherichia coli

The pst operon of Escherichia coli is composed of five genes that encode a high-affinity phosphate transport system. pst belongs to the PHO regulon, which is a group of genes and operons that are induced in response to phosphate limitation. The pst operon also has a regulatory role in the repression of PHO genes’ transcription under phosphate excess conditions. Transcription of pst is initiated at the promoter located upstream to the first gene, pstS. Immediately after its synthesis, the primary transcript of pst is cleaved into shorter mRNA molecules in a ribonuclease E-dependent manner. Other ribonucleases, such as RNase III and MazF, do not play a role in pst mRNA processing. RNase E is thus at least partially responsible for processing the pst primary transcript.

Alternative promoters in the pst operon of Escherichia coli

The pst operon of Escherichia coli is composed of five genes pstS, pstC, pstA, pstB and phoU, that encode a high-affinity phosphate transport system and a negative regulator of the PHO regulon. Transcription of pst is induced under phosphate shortage and is initiated at the promoter located upstream of the first gene of the operon, pstS. Here, we show by four different technical approaches the existence of additional internal promoters upstream of pstC, pstB and phoU. These promoters are not induced by Pi-limitation and do not possess PHO-box sequences. Plasmids carrying the pst internal genes partially complement chromosomal mutations in their corresponding genes, indicating that they are translated into functional proteins.

Stability of the pstS transcript of Escherichia coli

The pst operon of Escherichia coli is composed of five genes that encode a high-affinity phosphate transport system. As a member of the PHO regulon, pst transcription is activated under phosphate shortage conditions. Under phosphate-replete conditions, the pst operon also functions as a negative regulator of the PHO genes. Transcription of pst is initiated at the promoter located upstream to the first gene, pstS. Immediately after its synthesis, the primary transcript of pst is cleaved into shorter mRNA molecules. The transcription unit corresponding to pstS is significantly more abundant than the transcripts of the other pst genes due to stabilisation of pstS mRNA by a repetitive extragenic palindrome (REP) structure downstream to the pstS locus. The presence of the REP sequence also results in an increased level of PstS proteins. However, the surplus level of PstS proteins produced in the presence of REP does not contribute to the repressive role of Pst in PHO expression.