Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice

<p>Abstract</p> <p>Background</p> <p>Inducible conditional knockout animals are widely used to get insight in the function of genes and the pathogenesis of human diseases. These models frequently rely on Cre-mediated recombination of sequences flanked by Lox-P sites. To understand the consequences of gene disruption, it is essential to know the efficiency of the recombination process.</p> <p>Results</p> <p>Here, we describe a modification of the multiplex ligation-dependent probe amplification (MLPA), called extension-MLPA (eMLPA), which enables quantification of relatively small differences in DNA that are a consequence of Cre-mediated recombination. eMLPA, here applied on an inducible <it>Pkd1 </it>conditional deletion mouse model, simultaneously measures both the reduction of the floxed allele and the increase of the deletion allele in a single reaction thereby minimizing any type of experimental variation. Interestingly, with this method we were also able to observe the presence of the excised DNA fragment. This extra-chromosomal deletion-circle was detectable up to 5 months after activation of Cre.</p> <p>Conclusion</p> <p>eMLPA is a novel strategy which easily can be applied to measure the Cre-mediated recombination efficiency in each experimental case with high accuracy. In addition the fate of the deletion-circle can be followed simultaneously.</p

Effects of Pre- and Intermittent Warming of Cucumber Fruits on Chilling Injury, Titratable Acidity, Sugar and Ascorbic Acid Contents

1) 1℃及び5℃冷蔵のそれぞれのキュウリ果実に, 18℃, 24時間反復処理(2日おき3回)と, 36℃, 24時間処理(1℃では冷蔵前, また5℃では冷蔵3日後)とを行い, 低温障害並びに滴定酸度, 糖及びビタミンC含量の変化を調査した。2) 低温障害の抑制に関して, 1℃冷蔵においては36℃加温処理の著しい効果に対し, 18℃反復処理の効果はやや劣った。5℃冷蔵では両処理とも著しい抑制効果を示した。3) 滴定酸度, 糖含有量については, 1℃, 5℃いづれの冷蔵においても加温処理の影響は少なかった。4) 1℃及び5℃冷蔵によって, 還元型アスコルビン酸が減少し, 酸化型アスコルビン酸は増加した。ビタミンCの冷蔵による変化に対する抑制効果は, 1℃冷蔵の場合18℃反復処理では少なかったが, 36℃加温処理ではある程度認められた。5℃冷蔵においては両加温処理により, ビタミンCの変化は完全に抑制された。 / 1. Cucumber fruits were warmed at 18℃ for 24 hr three times of 2-day intervals during cold storage at 1 and 5℃, and also warmed at 36℃ for 24 hr before cold storage at 1℃ and after three days in cold storage at 4℃. Their effects on chilling injury, titratable acidity, sugar content and vitamin C content were determined. 2. Both warming treatments reduced chilling injury markedly for the fruit stored at 5℃. For the fruit stored at 1℃, the repeated warming at 18℃ produced only slight effect, but the warming at 36℃ pronounced effect. 3. Titratable acidity and sugar content were little affected by both treatments. 4. A reduction of L-ascorbic acid and an increase of dehydro-L-ascorbic acid during cold storage were completely prevented by both treatments for the fruit stored at 5℃. But there was slightly less effect for the fruit stored at 1℃

Characterization of the gene encoding human sarcolipin (SLN), a proteolipid associated with SERCA1: Absence of structural mutations in five patients with brody disease

Sarcolipin (SLN) is a low-molecular-weight protein that copurifies with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase (SERCA1). Genomic DNA and cDNA encoding human sarcolipin (SLN) were isolated and characterized and the SLN gene was mapped to chromosome 11q22-q23. Human, rabbit, and mouse cDNAs encode a protein of 31 amino acids. Homology of SLN with phospholamban (PLN) suggests that the first 7 hydrophilic amino acids are cytoplasmic, the next 19 hydrophobic amino acids form a single transmembrane helix, and the last 5 hydrophilic amino acids are lumenal. The cytoplasmic and transmembrane sequences are not well conserved among the three species, but the lumenal sequence is highly conserved. Like SERCA1, SLN is highly expressed in rabbit fast-twitch skeletal muscle, but it is expressed to a lower extent in slow-twitch muscle and to an even lower extent in cardiac muscle, where SERCA2a and PLN are highly expressed. It is expressed in only trace amounts in pancreas and prostate. SLN and PLN genes resemble each other in having two small exons, with their entire coding sequences lying in exon 2 and a large intron separating the two segments. Brody disease is an inherited disorder of skeletal muscle function, characterized by exercise-induced impairment of muscle relaxation. Mutations in the ATP2A1 gene encoding SERCA1 have been associated with the autosomal recessive inheritance of Brody disease in three families, but not with autosomal dominant inheritance of the disease. A search for mutations in the SLN gene in five Brody families, four of which were not linked to ATP2A1, did not reveal any alterations in coding, splice junction or promoter sequences. The homozygous deletion of C438 in the coding sequence of ATP2A1 in Brody disease family 3, leading to a frameshift and truncation following Pro147 in SERCA1, is the fourth ATP2A1 mutation to be associated with autosomal recessive Brody disease

Hereditary cancer registries improve the care of patients with a genetic predisposition to cancer:contributions from the Dutch Lynch syndrome registry

The Dutch Hereditary Cancer Registry was established in 1985 with the support of the Ministry of Health (VWS). The aims of the registry are: (1) to promote the identification of families with hereditary cancer, (2) to encourage the participation in surveillance programs of individuals at high risk, (3) to ensure the continuity of lifelong surveillance examinations, and (4) to promote research, in particular the improvement of surveillance protocols. During its early days the registry provided assistance with family investigations and the collection of medical data, and recommended surveillance when a family fulfilled specific diagnostic criteria. Since 2000 the registry has focused on family follow-up, and ensuring the quality of surveillance programs and appropriate clinical management. Since its founding, the registry has identified over 10,000 high-risk individuals with a diverse array of hereditary cancer syndromes. All were encouraged to participate in prevention programmes. The registry has published a number of studies that evaluated the outcome of surveillance protocols for colorectal cancer (CRC) in Lynch syndrome, as well as in familial colorectal cancer. In 2006, evaluation of the effect of registration and colonoscopic surveillance on the mortality rate associated with colorectal cancer (CRC) showed that the policy led to a substantial decrease in the mortality rate associated with CRC. Following discovery of MMR gene defects, the first predictive model that could select families for genetic testing was published by the Leiden group. In addition, over the years the registry has produced many cancer risk studies that have helped to develop appropriate surveillance protocols. Hereditary cancer registries in general, and the Lynch syndrome registry in particular, play an important role in improving the clinical management of affected families.</p

The Clinical Spectrum of Missense Mutations of the First Aspartic Acid of cbEGF-like Domains in Fibrillin-1 Including a Recessive Family

Marfan syndrome (MFS) is a dominant disorder with a recognizable phenotype. In most patients with the classical phenotype mutations are found in the fibrillin-1 gene (FBN1) on chromosome 15q21. It is thought that most mutations act in a dominant negative way or through haploinsufficiency. In 9 index cases referred for MFS we detected heterozygous missense mutations in FBN1 predicted to substitute the first aspartic acid of different calcium-binding Epidermal Growth Factor-like (cbEGF) fibrillin-1 domains. A similar mutation was found in homozygous state in 3 cases in a large consanguineous family. Heterozygous carriers of this mutation had no major skeletal, cardiovascular or ophthalmological features of MFS. In the literature 14 other heterozygous missense mutations are described leading to the substitution of the first aspartic acid of a cbEGF domain and resulting in a Marfan phenotype. Our data show that the phenotypic effect of aspartic acid substitutions in the first position of a cbEGF domain can range from asymptomatic to a severe neonatal phenotype. The recessive nature with reduced expression of FBN1 in one of the families suggests a threshold model combined with a mild functional defect of this specific mutation. © 2010 Wiley-Liss, Inc

A Dutch MYH7 founder mutation, p.(Asn1918Lys), is associated with early onset cardiomyopathy and congenital heart defects

Background Mutations in the myosin heavy chain 7 (MYH7) gene commonly cause cardiomyopathy but are less frequently associated with congenital heart defects. Methods In th

Experiences with array-based sequence capture; toward clinical applications

Although sequencing of a human genome gradually becomes an option, zooming in on the region of interest remains attractive and cost saving. We performed array-based sequence capture using 385K Roche NimbleGen, Inc. arrays to zoom in on the protein-coding and immediate intron-flanking sequences of 112 genes, potentially involved in mental retardation and congenital malformation. Captured material was sequenced using Illumina technology. A data analysis pipeline was built that detects sequence variants, positions them in relation to the gene, checks for presence in databases (eg, db single-nucleotide polymorphism (SNP)) and predicts the potential consequences at the level of RNA splicing and protein translation. In the samples analyzed, all known variants were reliably detected, including pathogenic variants from control cases and SNPs derived from array experiments. Although overall coverage varied considerably, it was reproducible per region and facilitated the detection of large deletions and duplications (copy number variations), including a partial deletion in the B3GALTL gene from a patient sample. For ultimate diagnostic application, overall results need to be improved. Future arrays should contain probes from both DNA strands, and to obtain a more even coverage, one could add fewer probes from densely and more probes from sparsely covered regions

Diagnostic exome sequencing in 266 Dutch patients with visual impairment

Inherited eye disorders have a large clinical and genetic heterogeneity, which makes genetic diagnosis cumbersome. An exome-sequencing approach was developed in which data analysis was divided into two steps: the vision gene panel and exome analysis. In the vision gene panel analysis, variants in genes known to cause inherited eye disorders were assessed for pathogenicity. If no causative variants were detected and when the patient consented, the entire exome data was analyzed. A total of 266 Dutch patients with different types of inherited eye disorders, including inherited retinal dystrophies, cataract, developmental eye disorders and optic atrophy, were investigated. In the vision gene panel analysis (likely), causative variants were detected in 49% and in the exome analysis in an additional 2% of the patients. The highest detection rate of (likely) causative variants was in patients with inherited retinal dystrophies, for instance a yield of 63% in patients with retinitis pigmentosa. In patients with developmental eye defects, cataract and optic atrophy, the detection rate was 50, 33 and 17%, respectively. An exome-sequencing approach enables a genetic diagnosis in patients with different types of inherited eye disorders using one test. The exome approach has the same detection rate as targeted panel sequencing tests, but offers a number of advantages. For instance, the vision gene panel can be frequently and easily updated with additional (novel) eye disorder genes. Determination of the genetic diagnosis improved the clinical diagnosis, regarding the assessment of the inheritance pattern as well as future disease perspective

The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently results in renal fallure due to progressive cyst development. The major locus, PKD1, maps to 16p13.3. We identified a chromosome translocation associated with ADPKD that disrupts a gene (PBP) encoding a 14 kb transcript in the PKD1 candidate region. Further mutations of the PBP gene were found in PKD1 patients, two deletions (one a de novo event) and a splicing defect, confirming that PBP is the PKD1 gene. This gene is located adjacent to the TSC2 locus in a genomic region that is reiterated more proximally on 16p. The duplicate area encodes three transcripts substantially homologous to the PKD1 transcript. Partial sequence analysis of the PKD1 transcript shows that it encodes a novel protein whose function is at present unknown