Screening of Living Kidney Donors for Genetic Diseases Using a Comprehensive Genetic Testing Strategy.

Related living kidney donors (LKDs) are at higher risk of end-stage renal disease (ESRD) compared with unrelated LKDs. A genetic panel was developed to screen 115 genes associated with renal diseases. We used this panel to screen six negative controls, four transplant candidates with presumed genetic renal disease and six related LKDs. After removing common variants, pathogenicity was predicted using six algorithms to score genetic variants based on conservation and function. All variants were evaluated in the context of patient phenotype and clinical data. We identified causal variants in three of the four transplant candidates. Two patients with a family history of autosomal dominant polycystic kidney disease segregated variants in PKD1. These findings excluded genetic risk in three of four relatives accepted as potential LKDs. A third patient with an atypical history for Alport syndrome had a splice site mutation in COL4A5. This pathogenic variant was excluded in a sibling accepted as an LKD. In another patient with a strong family history of ESRD, a negative genetic screen combined with negative comparative genomic hybridization in the recipient facilitated counseling of the related donor. This genetic renal disease panel will allow rapid, efficient and cost-effective evaluation of related LKDs.

High temperature is essential for preserved human sperm function during the devitrification process.

Sperm vitrification is a cryopreservation method based on high-speed freezing by direct exposure of cells in liquid nitrogen (N2L), thereby avoiding the traditional cooling curves of freezing. The objective of this work was to determine the optimal warming temperature for vitrified human spermatozoa in order to maintain their fertilisation potential. Spermatozoa were cryopreserved by direct plunging into N2L and warmed at different temperatures for 5 and 10 s at 38, 40 and 42 °C. Sperm motility was evaluated by the CASA system and the sperm membrane function by HOST test. It was detected that progressive motility of sperm warmed at 38, 40 and 42 °C was 26.4 ± 8.4%; 56.6 ± 16.3% and 65.4 ± 15%, respectively, with statistically significant differences between the temperatures of 38 and 40 °C and 38 and 42 °C (P < 0.05). The plasma membrane function evaluated by HOST test was better preserved at 42 °C (76.3 ± 2.0%) compared to 40 °C (43 ± 2%) and 38 °C (65.6 ± 1.5%). The temperature in the thawing process can affect the motility and plasma membrane integrity and function. The warming at 42 °C for thawed vitrified sperm is the optimum temperature to preserve the sperm physiological parameters.

Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study.

A collection of 1,108 case-parent trios ascertained through an isolated, nonsyndromic cleft lip with or without cleft palate (CL/P) was used to replicate the findings from a genome-wide association study (GWAS) conducted by Beaty et al. (Nat Genet 42:525-529, 2010), where four different genes/regions were identified as influencing risk to CL/P. Tagging SNPs for 33 different genes were genotyped (1,269 SNPs). All four of the genes originally identified as showing genome-wide significance (IRF6, ABCA4 and MAF, plus the 8q24 region) were confirmed in this independent sample of trios (who were primarily of European and Southeast Asian ancestry). In addition, eight genes classified as 'second tier' hits in the original study (PAX7, THADA, COL8A1/FILIP1L, DCAF4L2, GADD45G, NTN1, RBFOX3 and FOXE1) showed evidence of linkage and association in this replication sample. Meta-analysis between the original GWAS trios and these replication trios showed PAX7, COL8A1/FILIP1L and NTN1 achieved genome-wide significance. Tests for gene-environment interaction between these 33 genes and maternal smoking found evidence for interaction with two additional genes: GRID2 and ELAVL2 among European mothers (who had a higher rate of smoking than Asian mothers). Formal tests for gene-gene interaction (epistasis) failed to show evidence of statistical interaction in any simple fashion. This study confirms that many different genes influence risk to CL/P.

Temperature dependence on free volume in cured natural rubber and styrene-butadiene rubber blends.

A systematic study on the evolution of free volume as a function of the temperature in vulcanized at 433 K natural rubber (NR) and styrene butadiene rubber (SBR) in 25-75, 50-50, 75-25 NR-SBR (percent content of pure NR and SBR, respectively) blends was studied by positron annihilation lifetime spectroscopy. All samples were prepared with sulfur and TBBS (n-t-butyl-2-benzothiazole sulfenamide) as accelerator. The glass transition temperatures of the samples studied were determined by differential scanning calorimetry (DSC) and from lifetime data. In general, a sigmoidal-like complex behavior of the long-lived lifetime component, linked to the nanohole free volume, as a function of the temperature was found. For SBR, the slope of the ortho-positronium lifetime against temperature curves could be well-fitted using a linear function. For blends and also for NR, two different linear functions were necessary. This last behavior is explained in terms of the supercooled process involving a reconfiguration of the elastomeric chains. In the case of blends, the state of cure of NR and SBR in each NR-SBR sample was also taken into account in the discussion of the results obtained. Besides, thermal expansion coefficients of the free volumes in the transition and glassy region of all compounds were estimated. The differences observed in the values of this parameter are discussed by taking into account the morphology and formulation of each blend, the crosslink densities, and the role of the interphases formed between both NR and SBR elastomers.

Genetic studies in the Nigerian population implicate an MSX1 mutation in complex oral facial clefting disorders.

BACKGROUND: Orofacial clefts are the most common malformations of the head and neck, with a worldwide prevalence of 1 in 700 births. They are commonly divided into CL(P) and CP based on anatomic, genetic, and embryologic findings. A Nigerian craniofacial anomalies study (NigeriaCRAN) was set up in 2006 to investigate the role of gene-environment interaction in the origin of orofacial clefts in Nigeria. SUBJECTS AND METHODS: DNA isolated from saliva from Nigerian probands was used for genotype association studies and direct sequencing of cleft candidate genes: MSX1 , IRF6 , FOXE1, FGFR1 , FGFR2 , BMP4 , MAFB, ABCA4 , PAX7, and VAX1 , and the chromosome 8q region. RESULTS: A missense mutation A34G in MSX1 was observed in nine cases and four HapMap controls. No other apparent causative variations were identified. Deviation from Hardy Weinberg equilibrium (HWE) was observed in these cases (p = .00002). A significant difference was noted between the affected side for unilateral CL (p = .03) and bilateral clefts and between clefts on either side (p = .02). A significant gender difference was also observed for CP (p = .008). CONCLUSIONS: Replication of a mutation previously implicated in other populations suggests a role for the MSX1 A34G variant in the development of CL(P).

Identification of novel candidate genes associated with cleft lip and palate using array comparative genomic hybridisation.

AIM AND METHOD: We analysed DNA samples isolated from individuals born with cleft lip and cleft palate to identify deletions and duplications of candidate gene loci using array comparative genomic hybridisation (array-CGH). RESULTS: Of 83 syndromic cases analysed we identified one subject with a previously unknown 2.7 Mb deletion at 22q11.21 coinciding with the DiGeorge syndrome region. Eighteen of the syndromic cases had clinical features of Van der Woude syndrome and deletions were identified in five of these, all of which encompassed the interferon regulatory factor 6 (IRF6) gene. In a series of 104 non-syndromic cases we found one subject with a 3.2 Mb deletion at chromosome 6q25.1-25.2 and another with a 2.2 Mb deletion at 10q26.11-26.13. Analyses of parental DNA demonstrated that the two deletion cases at 22q11.21 and 6q25.1-25.2 were de novo, while the deletion of 10q26.11-26.13 was inherited from the mother, who also has a cleft lip. These deletions appear likely to be causally associated with the phenotypes of the subjects. Estrogen receptor 1 (ESR1) and fibroblast growth factor receptor 2 (FGFR2) genes from the 6q25.1-25.2 and 10q26.11-26.13, respectively, were identified as likely causative genes using a gene prioritization software. CONCLUSION: We have shown that array-CGH analysis of DNA samples derived from cleft lip and palate subjects is an efficient and productive method for identifying candidate chromosomal loci and genes, complementing traditional genetic mapping strategies.

Contributions of PTCH gene variants to isolated cleft lip and palate.

OBJECTIVE: Mutations in patched (PTCH) cause the nevoid basal cell carcinoma syndrome (NBCCS), or Gorlin syndrome. Nevoid basal cell carcinoma syndrome may present with developmental anomalies, including rib and craniofacial abnormalities, and predisposes to several tumor types, including basal cell carcinoma and medulloblastoma. Cleft palate is found in 4% of individuals with nevoid basal cell carcinoma syndrome. Because there might be specific sequence alterations in PTCH that limit expression to orofacial clefting, a genetic study of PTCH was undertaken in cases with cleft lip and/or palate (CL/P) known not to have nevoid basal cell carcinoma syndrome. RESULTS: Seven new normal variants spread along the entire gene and three missense mutations were found among cases with cleft lip and/or palate. One of these variants (P295S) was not found in any of 1188 control samples. A second variant was found in a case and also in 1 of 1119 controls. The third missense (S827G) was found in 5 of 1369 cases and in 5 of 1104 controls and is likely a rare normal variant. Linkage and linkage desequilibrium also was assessed using normal variants in and adjacent to the PTCH gene in 220 families (1776 individuals), each with two or more individuals with isolated clefting. Although no statistically significant evidence of linkage (multipoint HLOD peak = 2.36) was uncovered, there was borderline evidence of significant transmission distortion for one haplotype of two single nucleotide polymorphisms located within the PTCH gene (p = .08). CONCLUSION: Missense mutations in PTCH may be rare causes of isolated cleft lip and/or palate. An as yet unidentified variant near PTCH may act as a modifier of cleft lip and/or palate.