Identification of a new familial case of 3q29 deletion syndrome associated with cleft lip and palate via whole-exome sequencing.

Many unbalanced large copy number variants reviewed in the paper are associated with syndromic orofacial clefts, including a 1.6 Mb deletion on chromosome 3q29. The current report presents a new family with this recurrent deletion identified via whole-exome sequencing and confirmed by array comparative genomic hybridization. The proband exhibited a more severe clinical phenotype than his affected mother, comprising right-sided cleft lip/alveolus and cleft palate, advanced dental caries, heart defect, hypospadias, psychomotor, and speech delay, and an intellectual disability. Data analysis from the 3q29 registry revealed that the 3q29 deletion increases the risk of clefting by nearly 30-fold. No additional rare and pathogenic nucleotide variants were identified that could explain the clefting phenotype and observed intrafamilial phenotypic heterogeneity. These data suggest that the 3q29 deletion may be the primary risk factor for clefting, with additional genomic variants located outside the coding sequences, methylation changes, or environmental exposure serving as modifiers of this risk. Additional studies, including whole-genome sequencing or methylation analyses, should be performed to identify genetic factors underlying the phenotypic variation associated with the recurrent 3q29 deletion.

Identification of novel susceptibility genes for non-syndromic cleft lip with or without cleft palate using NGS-based multigene panel testing.

For non-syndromic cleft lip with or without cleft palate (ns-CL/P), the proportion of heritability explained by the known risk loci is estimated to be about 30% and is captured mainly by common variants identified in genome-wide association studies. To contribute to the explanation of the "missing heritability" problem for orofacial clefts, a candidate gene approach was taken to investigate the potential role of rare and private variants in the ns-CL/P risk. Using the next-generation sequencing technology, the coding sequence of a set of 423 candidate genes was analysed in 135 patients from the Polish population. After stringent multistage filtering, 37 rare coding and splicing variants of 28 genes were identified. 35% of these genetic alternations that may play a role of genetic modifiers influencing an individual's risk were detected in genes not previously associated with the ns-CL/P susceptibility, including COL11A1, COL17A1, DLX1, EFTUD2, FGF4, FGF8, FLNB, JAG1, NOTCH2, SHH, WNT5A and WNT9A. Significant enrichment of rare alleles in ns-CL/P patients compared with controls was also demonstrated for ARHGAP29, CHD7, COL17A1, FGF12, GAD1 and SATB2. In addition, analysis of panoramic radiographs of patients with identified predisposing variants may support the hypothesis of a common genetic link between orofacial clefts and dental abnormalities. In conclusion, our study has confirmed that rare coding variants might contribute to the genetic architecture of ns-CL/P. Since only single predisposing variants were identified in novel cleft susceptibility genes, future research will be required to confirm and fully understand their role in the aetiology of ns-CL/P.

PAX7 nucleotide variants and the risk of non-syndromic orofacial clefts in the Polish population.

OBJECTIVE: The etiology of non-syndromic cleft lip with or without cleft palate (nsCL/P) is multifactorial, heterogeneous, and still not completely understood. The aim of the present study was to examine the associations between common and rare PAX7 nucleotide variants and the risk of this common congenital anomaly in a Polish population. SUBJECTS AND METHODS: Eight top nsCL/P-associated PAX7 variants identified in our cleft genome-wide association study (GWAS) were selected for replication analysis in an independent group of patients and controls (n = 247 and n = 445, respectively). In addition, mutation screening of the PAX7 protein-coding region was conducted. RESULTS: Analysis of the pooled data from the GWAS and replication study confirmed that common PAX7 nucleotide variants are significantly associated with the increased risk of nsCL/P. The strongest individual variant was rs1339062 (c.586 + 15617T > C) with a p-value = 2.47E-05 (OR = 1.4, 95%CI: 1.20-1.64). Sequencing analysis identified a novel synonymous PAX7 substitution (c.87G > A, p.Val29Val) in a single patient with nsCLP. This transition located in the early exonic position was predicted to disrupt potential splice enhancer elements. CONCLUSION: Our study confirmed that PAX7 is a strong candidate gene for nsCL/P. Nucleotide variants of this gene contribute to the etiology of nsCL/P in the homogenous Polish population.

Identification of Novel Risk Variants of Non-Syndromic Cleft Palate by Targeted Gene Panel Sequencing.

Non-syndromic cleft palate (ns-CP) has a genetically heterogeneous aetiology. Numerous studies have suggested a crucial role of rare coding variants in characterizing the unrevealed component of genetic variation in ns-CP called the "missing heritability". Therefore, this study aimed to detect low-frequency variants that are implicated in ns-CP aetiology in the Polish population. For this purpose, coding regions of 423 genes associated with orofacial cleft anomalies and/or involved with facial development were screened in 38 ns-CP patients using the next-generation sequencing technology. After multistage selection and prioritisation, eight novel and four known rare variants that may influence an individual's risk of ns-CP were identified. Among detected alternations, seven were located in novel candidate genes for ns-CP, including COL17A1 (c.2435-1G>A), DLG1 (c.1586G>C, p.Glu562Asp), NHS (c.568G>C, p.Val190Leu-de novo variant), NOTCH2 (c.1997A>G, p.Tyr666Cys), TBX18 (c.647A>T, p.His225Leu), VAX1 (c.400G>A, p.Ala134Thr) and WNT5B (c.716G>T, p.Arg239Leu). The remaining risk variants were identified within genes previously linked to ns-CP, confirming their contribution to this anomaly. This list included ARHGAP29 (c.1706G>A, p.Arg569Gln), FLNB (c.3605A>G, Tyr1202Cys), IRF6 (224A>G, p.Asp75Gly-de novo variant), LRP6 (c.481C>A, p.Pro161Thr) and TP63 (c.353A>T, p.Asn118Ile). In summary, this study provides further insights into the genetic components contributing to ns-CP aetiology and identifies novel susceptibility genes for this craniofacial anomaly.

Chitosan-poly(ethylene oxide) nanofibrous mat as a vaginal platform for tenofovir disoproxyl fumarate - The effect of vaginal pH on drug carrier performance.

In the present work, a solution blow spun nanofibrous mat comprised of chitosan (CS) and poly(ethylene oxide) (PEO) was obtained as vaginal platform for tenofovir disoproxil fumarate (TDF) to prevent sexually transmitted infections. Apart from physicochemical and mechanical analysis, the specific steps involved studies on nanofibrous mat mucoadhesive and swelling characteristics upon pH fluctuations over the physiological range. Physicochemical analysis showed uniform drug distribution within the CS/PEO mat volume and pointed toward physical interactions between the drug and polymers. TDF-loaded CS/PEO nanofibrous mat was shown potentially safe when evaluated by the MTT metabolic activity and JC-1 assays in human vaginal epithelial cells VK2-E6/E7. In vitro antiviral studies indicated inhibition efficacy of TDF-CS/PEO nanofibrous mat toward HSV-2 virus and proved the SBS process does not change the microbicidal activity of drug molecule. Fluctuations in the physiological vaginal pH range of 3.8 to 5.0 substantially affected mucoadhesive and swelling behavior of chitosan which in turn impacted drug dissolution rate from polymer carrier. The rate of permeation and accumulation of TDF in vaginal tissue differed in response to vaginal pH. Faster drug permeation assessed at pH 5.0 suggests that an increase in vaginal pH could improve TDF bioavailability at earlier time points.

Novel Candidate Genes for Non-Syndromic Tooth Agenesis Identified Using Targeted Next-Generation Sequencing.

Non-syndromic tooth agenesis (ns-TA) is one of the most common dental anomalies characterized by the congenital absence of at least one permanent tooth (excluding third molars). Regarding the essential role of genetic factors in ns-TA aetiology, the present study aimed to identify novel pathogenic variants underlying hypodontia and oligodontia. In a group of 65 ns-TA patients and 127 healthy individuals from the genetically homogenous Polish population, the coding sequences of 423 candidate genes were screened using targeted next-generation sequencing. Pathogenic and likely pathogenic variants were identified in 37 (56.92%) patients, including eight nucleotide alternations of genes not previously implicated in ns-TA (CHD7, CREBBP, EVC, LEF1, ROR2, TBX22 and TP63). However, since only single variants were detected, future research is required to confirm and fully understand their role in the aetiology of ns-TA. Additionally, our results support the importance of already known ns-TA candidate genes (AXIN2, EDA, EDAR, IRF6, LAMA3, LRP6, MSX1, PAX9 and WNT10A) and provide additional evidence that ns-TA might be an oligogenic condition involving the cumulative effect of rare variants in two or more distinct genes.

Development of a Biphasic-Release Multiple-Unit Pellet System with Diclofenac Sodium Using Novel Calcium Phosphate-Based Starter Pellets.

Novel calcium phosphate-based starter pellets were used to develop a biphasic-release multiple-unit pellet system (MUPS) with diclofenac sodium as a model drug in the form of hard gelatin capsules. For comparative purposes, corresponding formulations based on the inert cores made of microcrystalline cellulose, sucrose and isomalt were prepared. The developed system consisted of two types of drug-layered pellets attaining different release patterns: delayed-release (enteric-coated) and extended-release. Dissolution characteristics were examined using both compendial and biorelevant methods, which reflected fed and fasting conditions. The results were collated with an equivalent commercial product but prepared with the direct pelletization technique.

CYP3A5 and CYP3A4, but not ABCB1 polymorphisms affect tacrolimus dose-adjusted trough concentrations in kidney transplant recipients.

BACKGROUND: Tacrolimus (TAC), acting as a calcineurin inhibitor, is an immunosuppressant widely used after kidney transplantation. TAC requires blood concentration monitoring due to large interindividual variability in its pharmacokinetics and a narrow therapeutic index. Since genetic factors are considered responsible for a part of the observed pharmacokinetic variability, hereby SNPs within the CYP3A4, CYP3A5 and ABCB1 genes in kidney transplant patients of Polish Caucasian origin were investigated. PATIENTS & METHODS: A total of 241 patients treated with TAC through the first year after kidney transplantation were genotyped for the presence of common SNPs: rs776746:A>G (CYP3A5*3), rs35599367:C>T (CYP3A4*22), rs2740574:A>G (CYP3A4*1B) and rs1045642:C>T (ABCB1 3435C>T) using TaqMan(®) assays. RESULTS: CYP3A5 expressers received significantly higher weight-adjusted TAC doses, and were characterized by markedly lower C0 and dose adjusted C0 values in the course of treatment. CYP3A4*1B was significantly associated with TAC pharmacokinetics in univariate analysis. Impact of the CYP3A4*22 allele was significant only at particular time points, that is, 3 months after transplantation, with marginal significance 6 months after transplantation. The ABCB1 genotype did not influence TAC pharmacokinetics. Multivariate analysis of all the studied loci demonstrated that only the CYP3A5*1 (starting from month 1) and CYP3A4*22 alleles (at 3 and 6 months) were independent predictors of TAC dose-adjusted C0. CONCLUSION: Our results confirm the impact of the CYP3A4*22 allele on TAC pharmacokinetics, as a second significant genetic factor (in addition to the CYP3A5*1 allele) influencing TAC dose-adjusted blood concentrations in kidney transplant recipients.

[Transient hyponatremia the first symptome of multiple endocrine neoplasia type 1 (MEN 1)--case report]. / Przejsciowa hiponatremia pierwszym objawem zespolu mnogiej gruczolakowatosci typu 1 (MEN 1)--przypadek kliniczny.

Transient character of laboratory abnormalities is usually explained as a false result of estimation. Spontaneous disappearance of laboratory abnormalities could be recognized as non-important and contradicting serious disease. Presented case of multiple endocrine neoplasia type 1 (MEN 1) denies these assumptions. Sixty-nine-years old woma was admitted to the internal department because of weakness, fever, dehydration and hypotension--symptom accompanying an upper respiratory infection. Moderate hyponatremia (122.6 mmol/l) was found in a routine laborator examination. All clinical symptoms as well as hyponatremia disappeared during hospitalization. Transient hyponatremi was not ignored. Further laboratory and hormonal investigations were performed. Fasting, morning serum cortisol concentration and 24-hours urinary excretion of free corticosterids were decreased and Synacten stimulation test reveale incomplete cortisol stimulation. Pituitary tumour was found in the CT scan. Moreover blood ionized calcium an parathyroid hormone were elevated. These results revealed secondary adrenal insufficiency, non-functioning pituitary tumor and hyperparathyroidism. Removal of pituitary adenoma was done. Asymptomatic hypercalcemia persists. Presented MEN 1 was atypical because: 1. Usually hyperparathyroidism is the first and most frequent symptom while in our patient the first symptom was transient hyponatremia secondary to the pituitary tumor, 2. was diagnosed in the old age while the majority of MEN 1 patients are younger, 3. asymptomatic course of hypercalcemia indicates that in some of elder patients removal of the parathyroid glands might not be necessary.

[Effectiveness of iodine prophylaxis in hypertensive patients on salt restricted diet]. / Efektywnosc profilaktyki jodowej w warunkach ograniczonego spozycia soli kuchennej u chorych z nadcisnieniem tetniczym.

BACKGROUND: Iodine prophylaxis in Poland started in 1935 however it has been interrupted during Second World War, and also in 1980. In January 1997 a new approach of preventing iodine deficiency was introduced as obligatory iodination model of household salt to be extent of 30 mg KI/kg. The aim of the study was to determine iodine intake after introduction of mandatory salt iodization in group of persons being on low salt diet because of hypertension. Investigated group and methods: The study was performed on 227 persons (94 males and 133 females), aged 18-89 years (average age 56 +/- 15,2 years). Each person answered questions concerning salt consumption and other products being additional source of iodine (fishes, pharmaceutical preparations containing iodine). Subjects were divided into two groups: according to the amount of salt consumption (119 persons restricted to household salt and 108 persons used kitchen salt) and according to the value of blood pressure (170 persons was hypertensive and 57 persons was normotensive). The urine iodine concentration (UIC) was determined in morning urine samples by the method of Sandell and Kolthoff. RESULTS: The average UIC in the whole studied group was 156,1 +/- 124,9 microg/l. Woman showed lower UIC than man (132,9 microg/l vs 177,5 microg/l). In 8% of patients iodine concentration was below 50 microg/l and in 64% of patients it was 100 microg/l or more. In the case of persons consuming limited, low amount of salt, the average urine iodine concentration was 150,3 microg/l, whereas those who add some salt to a dish was 162,4 microg/l. In hypertension patients ioduria was lower than in persons with normal blood pressure (153,1 microg/l vs 164,9 microg/l). CONCLUSIONS: The model of iodine prophylaxis has used in Poland is effective. It also concerns persons consuming limited amount of salt in the our studies group. The amount of consumed salt should be determined by measuring of Na+ concentration in 24-hour urine collection. Additional monitoring of iodine supply is necessary because of possible consumption of iodine from other sources than salt.