Spatial Multi-omics Reveals the Role of the Wnt Modulator, Dkk2, in Palatogenesis'.

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which is the most common of the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study used the Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. Although prior research has identified the upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 in the mesenchyme. Moreover, the loss of Pax9 leads to a disruption in the normal osteodifferentiaion of palatal osteogenic mesenchymal cells. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

Variations in maternal adenylate cyclase genes are associated with congenital Zika syndrome in a cohort from Northeast, Brazil.

BACKGROUND: Vertical transmission of Zika virus (ZIKV) is associated with congenital malformations but the mechanism of pathogenesis remains unclear. Although host genetics appear to play a role, no genetic association study has yet been performed to evaluate this question. In order to investigate if maternal genetic variation is associated with Congenital Zika Syndrome (CZS), we conducted a case-control study in a cohort of Brazilian women infected with ZIKV during pregnancy. METHODS: A total of 100 women who reported symptoms of zika during pregnancy were enrolled and tested for ZIKV. Among 52 women positive for ZIKV infection, 28 were classified as cases and 24 as controls based on the presence or absence of CZS in their infants. Variations in the coding region of 205 candidate genes involved in cAMP signaling or immune response were assessed by high throughput sequencing and tested for association with development of CZS. RESULTS: From the 817 single nucleotide variations (SNVs) included in association analyses, 22 SNVs in 17 genes were associated with CZS under an additive model (alpha = 0.05). Variations c.319T>C (rs11676272) and c.1297G>A, located at ADCY3 and ADCY7 genes showed the most prominent effect. The association of ADCY3 and ADCY7 genes was confirmed using a Sequence Kernel Association Test to assess the joint effect of common and rare variations, and results were statistically significant after adjustment for multiple comparisons (P < 0.002). CONCLUSION: These results suggest that maternal ADCY genes contribute to ZIKV pathogenicity and influence the outcome of CZS, being promising candidates for further replication studies and functional analysis.

Congenital bilateral absence of the vas deferens as an atypical form of cystic fibrosis: reproductive implications and genetic counseling.

Congenital bilateral absence of the vas deferens (CBAVD) is found in 1% to 2% of males with infertility and is present in 6% of obstructive azoospermia cases. Nearly 95% of men with cystic fibrosis (CF, an autosomal recessive disorder) have CBAVD. There are genetic links between CBAVD and CF. Some mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) can lead to CBAVD as a monosymptomatic form of CF. With the use of assisted reproductive techniques (ART), especially testicular or epididymal sperm aspiration, intracytoplasmic sperm injection, and in vitro fertilization, it is possible that men with CBAVD can produce offspring. Therefore, genetic counseling should be offered to couples undergoing ART to discuss the probability of having offspring that carry CFTR gene mutations. The aim of this review was to present the main cause of CBAVD, to call attention to its implications for assisted reproduction, and to show the importance of genetic counseling for couples where men have CBAVD, as they can have offspring with a lethal disease.

Age-dependent effects of Armc5 haploinsufficiency on adrenocortical function.

Inactivating mutations in the Armadillo repeat-containing 5 (ARMC5) gene have recently been discovered in primary macronodular adrenal hyperplasia (PMAH), a cause of Cushing syndrome. Biallelic ARMC5 inactivation in PMAH suggested that ARMC5 may have tumor suppressor functions in the adrenal cortex. We generated and characterized a new mouse model of Armc5 deficiency. Almost all Armc5 knockout mice died during early embryonic development, around 6.5 and 8.5 days. Knockout embryos did not undergo gastrulation, as demonstrated by the absence of mesoderm development at E7.5. Armc5 heterozygote mice (Armc5+/-) developed normally but at the age of 1 year, their corticosterone levels decreased; this was associated with a decrease of protein kinase A (PKA) catalytic subunit α (Cα) expression both at the RNA and protein levels that were also seen in human patients with PMAH and ARMC5 defects. However, this was transient, as corticosterone levels normalized later, followed by the development of hypercorticosteronemia in one-third of the mice at 18 months of age, which was associated with increases in PKA and Cα expression. Adrenocortical tissue analysis from Armc5+/- mice at 18 months showed an abnormal activation of the Wnt/ß-catenin signaling pathway in a subset of zona fasciculata cells. These data confirm that Armc5 plays an important role in early mouse embryonic development. Our new mouse line can be used to study tissue-specific effects of Armc5. Finally, Armc5 haploinsufficiency leads to Cushing syndrome in mice, but only later in life, and this involves PKA, its catalytic subunit Cα, and the Wnt/ß-catenin pathway.

Cystic fibrosis in Afro-Brazilians: XK haplotypes analysis supports the European origin of p.F508del mutation.

Cystic fibrosis (CF) is a common autosomal recessive disorder, being the p.F508del the most frequent mutation. Also, a nearby restriction fragment length polymorphism (RFLP) named XK (KM19 and XV2C) is non-randomly associated with specific CF alleles. Our aim was to analyze the occurrence of the p.F508del mutation and XK haplotypes in Afro-Brazilians CF patients and controls, since these data is available for the other two main ethnic groups found in Brazil (Euro-Brazilians and Brazilian Amerindians), contributing for the whole comprehension of these haplotypes in the Brazilian population. A total of 103 patients and 54 controls were studied. PCR and PCR-RFLP methodologies were used to identify the presence of the p.F508del and the XK haplotype in the subjects. The combined data show that 84.2% of p.F508del mutation is associated with haplotype B and only 15.8% with haplotype A; no other haplotypes were found to be associated with this mutation. Our data suggest that the occurrence of p.F508del mutation and haplotype B in Afro-Brazilian patients occurs probably due to admixture with Euro-descendants. Therefore this mutation and haplotype could be used as a admixture marker.

SDH Subunit Mutation Status in Saliva: Genetic Testing in Patients with Pheochromocytoma.

Germline mutations occur in up to 30-40% of pheochromocytoma/paraganglioma, with mutations in the succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD) being the most common. Blood samples are favored for obtaining high quality DNA, however, leukocytes can also be obtained by collecting saliva. The aim of this study was to determine whether SDHB and SDHD gene mutations in patients with pheochromocytoma/paraganglioma could be determined using a salivary sample. Paired blood and salivary samples were collected from 30 patients: 9 SDHB mutation positive, 13 with a SDHD mutation, and 8 without any SDHx mutations. The Oragene DISCOVER kit was used to collect and extract DNA from saliva. Blood DNA was extracted from EDTA blood samples. The DNA purification and concentration were measured by spectrophotometry. The 8 exons of SDHB and the 4 exons of SDHD were amplified and sequenced by PCR-based bidirectional Sanger sequencing. Total DNA yields from blood DNA were similar to those obtained from saliva DNA [mean (±SD) saliva vs. blood DNA concentration 514.6 (±580.8) ng/µl vs. 360.9 (±262.7) ng/µl; p=0.2)]. The purity of the saliva DNA samples was lower than that of blood [mean OD260/OD280 ratio 1.78 (±0.13) vs. 1.87 (±0.04); p=0.001, respectively], indicating more protein contamination in the saliva-extracted DNA. This study shows that salivary DNA collected from patients with pheochromocytoma/paraganglioma is a good alternative for extraction of genomic DNA for its high DNA concentration and acceptable purity and can be used as an alternative to blood derived DNA in screening for SDHB and SDHD mutations.

Functional screen analysis reveals miR-26b and miR-128 as central regulators of pituitary somatomammotrophic tumor growth through activation of the PTEN-AKT pathway.

MicroRNAs (miRNAs) have been involved in the pathogenesis of different types of cancer; however, their function in pituitary tumorigenesis remains poorly understood. Cyclic-AMP-dependent protein kinase-defective pituitaries occasionally form aggressive growth-hormone (GH)-producing pituitary tumors in the background of hyperplasia caused by haploinsufficiency of the protein kinase's main regulatory subunit, PRKAR1A. The molecular basis for this development remains unknown. We have identified a 17-miRNA signature of pituitary tumors formed in the background of hyperplasia (caused in half of the cases by PRKAR1A-mutations). We selected two miRNAs on the basis of their functional screen analysis: inhibition of miR-26b expression and upregulation of miR-128 suppressed the colony formation ability and invasiveness of pituitary tumor cells. Furthermore, we identified that miR-26b and miR-128 affected pituitary tumor cell behavior through regulation of their direct targets, PTEN and BMI1, respectively. In addition, we found that miR-128 through BMI1 direct binding on the PTEN promoter affected PTEN expression levels and AKT activity in the pituitary tumor cells. Our in vivo data revealed that inhibition of miR-26b and overexpression of miR-128 could suppress pituitary GH3 tumor growth in xenografts. Taken together, we have identified a miRNA signature for GH-producing pituitary tumors and found that miR-26b and miR-128 regulate the activity of the PTEN-AKT pathway in these tumors. This is the first suggestion of the possible involvement of miRNAs regulating the PTEN-AKT pathway in GH-producing pituitary tumor formation in the context of hyperplasia or due to germline PRKAR1A defects. MiR-26b suppression and miR-128 upregulation could have therapeutic potential in GH-producing pituitary tumor patients.

Expression of pancreatic endocrine markers by mesenchymal stem cells from human umbilical cord vein.

BACKGROUND: Mesenchymal stem cells (MSCs) from human umbilical cord vein have great potential for use in cell therapy because of their ease of isolation, expansion, and differentiation, in addition to their relative acceptance from the ethical point of view. Obtaining the umbilical cord at birth does not present any risk to either mother or child. OBJECTIVE: To isolate and promote in vitro expansion and differentiation of MSCs from human umbilical cord vein into cells with a pancreatic endocrine phenotype. METHODS: Mesenchymal stem cells obtained from human umbilical cord vein via collagenase digestion were characterized at cytochemistry and fluorescent-activated cell sorting, and expanded in vitro. Differentiation of MSCs into an endocrine phenotype was induced using high-glucose (23 mmol/L) medium containing nicotinamide, exendin-4, and 2-mercaptoethanol. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was analyzed using immunofluorescence. RESULTS: Cells isolated from the umbilical cord vein were MSCs as confirmed at cytochemistry and fluorescent-activated cell sorting. Expression of somatostatin, glucagon, and pancreatic and duodenal homeobox 1 by differentiated cells was demonstrated using immunofluorescence. Insulin was not expressed. CONCLUSIONS: The MSC differentiation protocol used in the present study induced expression of some endocrine markers. Insulin was not produced by these cells, probably because of incomplete induction of differentiation.

Cystic fibrosis gene variability in two southern Brazilian Amerindian populations: analysis of the deltaF508 mutation and the KM19 and XV2C haplotypes.

The frequencies of the deltaF508 deletion, the most common cystic fibrosis mutation in Europeans and European-derived populations, and the XV2C and KM19 restriction fragment length polymorphisms that are tightly linked to the CFTR locus vary among populations. To determine the distribution of these extragenic markers and of the deltaF508 mutation, we analyzed 326 chromosomes of individuals from two South American Indian populations, the Guarani and the Kaingang. The allele and haplotype frequencies differed greatly between the two populations as well as among Amerindians and normal European Brazilians and European Brazilian cystic fibrosis patients. The absence of the deltaF508 mutation and the B haplotype are in agreement with the hypothesis that the deltaF508 mutation occurred after the divergence of these two populations. This finding is useful for populations containing a large Amerindian component and helps us to understand the origins of the deltaF508 deletion, the most common cystic fibrosis mutation in Europeans and European-derived populations, as well as the different incidences of cystic fibrosis in continental groups.

Cystic fibrosis in a southern Brazilian population: characteristics of 90% of the alleles.

Cystic fibrosis (CF) is a genetic disease that frequently leads to death in infancy among Europeans and their descendants. The goals of the present study were to analyze the molecular aspects of CFTR gene characterizing mutations, their frequencies, and the haplotypes formed by four CFTR gene intragenic markers, IVS8-6(T)n, IVS8CA, IVS17bTA and IVS17bCA, in a southern Brazilian population of Caucasian origin. DNA samples from 56 non-related CF patients were analyzed using scanning techniques (single strand conformation polymorphism and denaturing gradient gel electrophoresis), restriction fragment length polymorphism and direct DNA sequencing to identify the mutations. Our results revealed a total of 25 different CF mutations representing nearly 90% of CF alleles, two being novel mutations. Microsatellite haplotypes were defined for CF and normal alleles. The mutational spectrum and the associated haplotypes described for the first time in this study should prove relevant for genetic counselling and CF population screening in Brazil. Moreover, our results suggest the presence of a major Mediterranean component in the contemporary Brazilian CF patient pool.

Polymorphism of LMP2, TAP1, LMP7 and TAP2 in Brazilian Amerindians and Caucasoids: implications for the evolution of allelic and haplotypic diversity.

In the class II region of the major histocompatibility complex (MHC), four genes implicated in processing of MHC class I-presented antigens have been described. Two of these (TAP1 and TAP2) code for endoplasmic reticulum membrane transporter proteins and the other two (LMP2 and LMP7) for proteasome subunits. These genes are polymorphic, although much less so than classical MHC class I and II genes. There is controversy concerning the possible functional implications of this variation. Population genetics is one of the means of investigating the evolutionary and functional significance of genetic polymorphisms; however, few populations have been analysed with respect to TAP and LMP diversity. We present here the polymorphism of TAP1, TAP2, LMP2 and LMP7 genes in the Kaingang and Guarani Amerindian tribes, and in the Caucasoid population of the Brazilian State of Paraná. Allele frequencies found in the Caucasoids were close to those described for similar populations. Amerindians had a somewhat more restricted polymorphism, and allele and haplotype frequencies differed greatly between the two tribes. Overall linkage disequilibrium (LD) between the four genes was low in the Caucasoids, but high in the Amerindians, for which significant LD was seen for all informative pairs of loci. Comparing results of this and previous studies we observed that, whenever significant LD occurs in non-Amerindians, it tends to be similar in the different ethnic groups. While this might be interpreted as evidence of co-evolution of genes in the TAP-LMP region, the high haplotypic diversity in all populations and low LD in non-Amerindians indicate absence of co-evolution of the different genes. Distributions of allele and genotype frequencies are consistent with the hypothesis of selective neutrality. We conclude that genetic polymorphism of the human TAP and LMP genes and haplotypes is of little, if any, functional significance.

HLA-DQA1 and HLA-DQB1 alleles and haplotypes in two Brazilian Indian tribes: evidence of conservative evolution of HLA-DQ.

Nucleotide sequence polymorphism of the HLA-DQA1 and HLA-DQB1 class II genes was analyzed in the Kaingang and Guarani Amerindians from southern Brazil using PCR sequence-specific oligonucleotide typing methods. Four different DQA1-DQB1 haplotypes were found: DQA1*0401-DQB1*0402 (associated with DRB1*0802, DRB1*08041, and DRB1*0807), DQA1*0501-DQB1*0301 (associated with DRB1*1602, DRB1*1413, and DRB1*1402), DQA1*03-DQB1*0302 (associated with DRB1*0404 and DRB1*0411), and DQA1*03-DQB1*03032 (associated with DRB1*09012). These HLA-DQA1 and HLA-DQB1 alleles and haplotypes are common in many other populations of all major ethnic groups. Alleles and haplotypes introduced into the populations by post-Columbian admixture were seen at low frequency both in the Kaingang (3.2%) and in the Guarani (3.8%). No novel HLA-DQA1 and HLA-DQB1 alleles have thus far been identified in Amerindians. This differs from previous results for HLA-DRB1, another class II locus presenting novel alleles (i.e., alleles not found in other ethnic groups and probably generated after migration of paleo-Indians to the Americas) in the Guarani and in other South American Indian populations. The distribution of the HLA-DQ alleles and haplotypes in Amerindians indicates a weaker diversifying selective pressure on the HLA-DQ genes compared with HLA-DRB1 and HLA-B. The more conservative evolution of HLA-DQA1 and HLA-DQB1 compared with HLA-DRB1 is strong evidence of (still not well-defined) functional differences of these class II genes.