https://aasldpubs.onlinelibrary.wiley.com/doi/pdf/10.1002/hep.20704

AASLD practice guidelines: Evaluation of the patient for liver transplantation

https://aasldpubs.onlinelibrary.wiley.com/doi/pdf/10.1002/hep.26972

Evaluation for liver transplantation in adults: 2013 practice guideline by the American Association for the Study of Liver Diseases and the American Society of Transplantation

Serum metabolite concentrations provide a direct readout of biological processes in the human body, and they are associated with disorders such as cardiovascular and metabolic diseases. We present a genome-wide association study (GWAS) of 163 metabolic traits measured in human blood from 1,809 participants from the KORA population, with replication in 422 participants of the TwinsUK cohort. For eight out of nine replicated loci (FADS1, ELOVL2, ACADS, ACADM, ACADL, SPTLC3, ETFDH and SLC16A9), the genetic variant is located in or near genes encoding enzymes or solute carriers whose functions match the associating metabolic traits. In our study, the use of metabolite concentration ratios as proxies for enzymatic reaction rates reduced the variance and yielded robust statistical associations with P values ranging from 3 x 10(-24) to 6.5 x 10(-179). These loci explained 5.6%-36.3% of the observed variance in metabolite concentrations. For several loci, associations with clinically relevant parameters have been reported previously.

/pdf/a-genome-wide-perspective-of-genetic-variation-in-human-2ikckemxzx.pdf

A genome-wide perspective of genetic variation in human metabolism

Trinucleotide repeats that expand in human disease form hairpin structures in vitro

Repeated elements can be widely abundant in eukaryotic genomes, composing more than 50% of the human genome, for example. It is possible to classify repeated sequences into two large families, "tandem repeats" and "dispersed repeats." Each of these two families can be itself divided into subfamilies. Dispersed repeats contain transposons, tRNA genes, and gene paralogues, whereas tandem repeats contain gene tandems, ribosomal DNA repeat arrays, and satellite DNA, itself subdivided into satellites, minisatellites, and microsatellites. Remarkably, the molecular mechanisms that create and propagate dispersed and tandem repeats are specific to each class and usually do not overlap. In the present review, we have chosen in the first section to describe the nature and distribution of dispersed and tandem repeats in eukaryotic genomes in the light of complete (or nearly complete) available genome sequences. In the second part, we focus on the molecular mechanisms responsible for the fast evolution of two specific classes of tandem repeats: minisatellites and microsatellites. Given that a growing number of human neurological disorders involve the expansion of a particular class of microsatellites, called trinucleotide repeats, a large part of the recent experimental work on microsatellites has focused on these particular repeats, and thus we also review the current knowledge in this area. Finally, we propose a unified definition for mini- and microsatellites that takes into account their biological properties and try to point out new directions that should be explored in a near future on our road to understanding the genetics of repeated sequences.

/pdf/comparative-genomics-and-molecular-dynamics-of-dna-repeats-39tmmpiitw.pdf

Comparative Genomics and Molecular Dynamics of DNA Repeats in Eukaryotes

This study addresses mechanism of instability of the FMR-1 (CGG)n-repeat, and investigates features which may distinguish between normal stable and fragile X unstable repeats. To achieve this, we have sequenced 178 alleles to analyze patterns of AGG interruptions within the CGG repeat, and have typed the (CA)n-repeat at DXS548 for 204 chromosomes. Overall, our data is consistent with the idea that the length of uninterrupted CGG repeats determines instability. We predict that certain sequence configurations [no AGG, and (CGG)9-11AGG(CGG) > or = 20] present in the general population, are predisposed towards replication slippage. Association between these proposed predisposing repeats and DXS548 alleles may explain the previously reported frequencies of fragile X mutations and large-size normal repeats on specific haplotype backgrounds. We propose that predisposing alleles arise in the general population by as yet undefined mechanism(s) which introduce a relatively long stretch of pure CGG repeat at the 3'-end (relative to the direction of transcription) of the FMR-1 repeat region. The 3' pure repeat may then be susceptible to further expansion by replication slippage. Slippage on these predisposing chromosomes could accumulate over many generations until a threshold size is reached, at which point the repeat is susceptible to greater instability (i.e. premutation stage). Thus, results suggest that evolution of fragile X full mutations could involve 4 definable stages: 1) ancestral events leading to the formation of predisposing alleles which have large total repeat length (e.g. between 35 to 50) but no AGG or 1 AGG; 2) gradual slippage of these predisposing alleles to small premutations (S alleles); 3) conversion from S alleles to larger premutations (Z); 4) massive expansion from a Z allele to a full mutation (L).

Sequence analysis of the fragile X trinucleotide repeat: implications for the origin of the fragile X mutation

https://aasldpubs.onlinelibrary.wiley.com/doi/pdf/10.1002/hep.510310227

Iron overload in cirrhosis : HFE genotypes and outcome after liver transplantation

The frequency of short-chain acyl-CoA dehydrogenase gene variants in the US population and correlation with the C4-acylcarnitine concentration in newborn blood spots

Background: Multiple endocrine neoplasia type 2 (MEN2) is a cancer syndrome with well-characterized causative mutations. Missense mutations in ∼15 codons of the RET gene have been linked to disease phenotypes in the vast majority of cases. These missense mutations range from very simple single nucleotide base changes to more numerous changes at a given codon; they therefore are often tested for by more than one DNA-based diagnostic method. We developed and evaluated a Pyrosequencing™ technology-based approach for MEN2 mutation testing that allows both simple and complex mutations to be analyzed on one platform.

Methods: Archived DNA from peripheral blood of patients referred to the Mayo Clinic Molecular Genetics laboratory for MEN2 testing was selected. One to all of codons 609, 611, 618, 620, 630, 634, 768, 804, and 918 were analyzed by Pyrosequencing technology to match the original analysis of each patient. Template PCRs were set up using an automated liquid handler; the subsequent post-PCR preparation step was performed manually, and the sequencing was performed by a PSQ 96 instrument. Samples were tested in batch sizes expected to occur routinely.

Results: We analyzed samples from 217 patients who previously tested negative for MEN2 and 230 patients who previously tested positive, for a total of 1449 sequencing reactions. One discrepant result was found (100% concordant for negatives and 99.6% concordant for positives). A total of 37 unique mutations or alterations of unknown significance were analyzed.

Conclusion: Pyrosequencing technology offers an accurate, nonisotopic, simple, and rapid method for the analysis of DNA from patients suspected of having MEN2.

/pdf/pyrosequencing-technology-as-a-method-for-the-diagnosis-of-4icyyr0ov0.pdf

Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2

During the last decade, research and clinical use of real-time PCR applications has continued to grow in importance (1). Many laboratories that use real-time PCR with fluorescent probes experience an unexplained loss of probe fluorescence at some stage, in particular with pairs of fluorescence resonance energy transfer (FRET) probes. Photobleaching is often assumed to be the cause. Structural integrity of the oligonucleotides is also a major factor, and its loss has been shown to correlate with repeated freeze–thaw cycles (2). Laboratories guard against these two problems by aliquoting probes and protecting them from light. Despite these precautions, inexplicable FRET probe failures are still observed. In one recent such case, we were able to determine an additional mechanism for FRET probe failure: loss of the phosphate cap from the 3′ end of a probe. To our knowledge, this has not been described previously. Our studies revealed that this may be a common and important problem, intrinsic to 3′-phosphate-blocking chemistry. We also found that alternative terminating groups may be a preferable option to 3′-phosphate blocking.

A 3-nmol/L synthesis-scale LightCycler™ hybridization probe set was purchased from Idaho Technology Inc. Biochem in April 2003. As is common practice, the manufacturer produced a large-scale synthesis and, after shipping our order, archived the remainder for a possible future reorder. The first half of the batch (α-probe set) was sent immediately, whereas the second half (β-probe set) was stored lyophilized for 6 months at −20 °C and then shipped with our next order.

Oligonucleotides from the first shipment were used in PCR reactions in a LightCycler with satisfactory results. PCR conditions were as follows: 1× LightCycler FastStart DNA Master Hybridization Probe Mix (Roche Diagnostics), MgCl2 (final concentration, 3.5 mM), 0.5 μM each of the forward (5′-GGCCTTTCTGAAGCAAG-3′) and reverse (5′-GACGATTTCTTATTTCACAGCTCC-3′) primers, 0.2 μM each of the donor …

/pdf/substitution-of-3-phosphate-cap-with-a-carbon-based-blocker-4bphiwgcis.pdf

Substitution of 3′-Phosphate Cap with a Carbon-Based Blocker Reduces the Possibility of Fluorescence Resonance Energy Transfer Probe Failure in Real-Time PCR Assays

Kent E. Kruckeberg

Papers

Sequence analysis of the fragile X trinucleotide repeat: implications for the origin of the fragile X mutation

Iron overload in cirrhosis : HFE genotypes and outcome after liver transplantation

The frequency of short-chain acyl-CoA dehydrogenase gene variants in the US population and correlation with the C4-acylcarnitine concentration in newborn blood spots

Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2

Substitution of 3′-Phosphate Cap with a Carbon-Based Blocker Reduces the Possibility of Fluorescence Resonance Energy Transfer Probe Failure in Real-Time PCR Assays