Mind the model for end-stage liver disease: model for end-stage liver disease score as an indicator of hemoderivate transfusion and survival in liver transplantation.

The scarcity of liver donors has increased the necessity to closely select recipients to improve liver transplantation outcomes. If we were able to recognize those recipients with the best outcomes, then this could result in a better and more accurate selection of our donors. Hemoderivate transfusion is one of the main important factors to analyse. We reviewed the data of all of our liver transplant recipients from May 1998 to December 2013 and selected 888 patients with complete records. We divided these patients into 5 groups to get a better selection. We found differences between these groups with respect to the following: recipient age at the time of transplantation, percentage of patients with hepatocarcinoma, and those with hepatitis C virus (HCV)-related etiology. Also, intensive care unit (ICU) time and the need for retransplantation were distinctive factors with observable differences between our groups. With respect to model for end-stage liver disease (MELD) score, the groups were clearly defined by their mean MELD score, finding significant statistical differences between these groups with respect to this score. We also found a significant relationship between MELD scores and survival in the different groups. This is also the first review in which the MELD score and intraoperative transfusion requirements are well associated with patient and graft survival.

Construction of small-insert libraries enriched for short tandem repeat sequences by marker selection.

These protocols construct a representative small-insert genomic DNA library in a phagemid vector. First, size-selected DNA fragments are ligated into a phagemid vector. In the second protocol, the resulting small-insert phagemid library is propagated in a bacterial strain combining mutations at the dut and ung loci, which permit incorporation of uracil in place of thymidine during DNA replication. Infection of the phagemid library with M13 helper phage permits recovery of this library as single-stranded DNA (ssDNA). Finally, this uracil-substituted ssDNA is used as a template for primer extension using an oligonucleotide whose sequence corresponds to the STR class of interest [e.g., (GATA)10] as primer. The products of this primer-extension reaction are transformed into an E. coli strain maintaining wild-type genes at the dut and ung loci. Under these conditions, uracil-substituted ssDNA will be restricted from growing by the host-encoded uracil-N-glycosylase, while the primer-extended products are capable of replicating.

Characterization of Alu repeats that are associated with trinucleotide and tetranucleotide repeat microsatellites.

The association of subclasses of Alu repetitive elements with various classes of trinucleotide and tetranucleotide microsatellites was characterized as a first step toward advancing our understanding of the evolution of microsatellite repeats. In addition, information regarding the association of specific classes of microsatellites with families of Alu elements was used to facilitate the development of genetic markers. Sequences containing Alu repeats were eliminated because unique primers could not be designed. Various classes of microsatellites are associated with different classes of Alu repeats. Very abundant and poly(A)-rich microsatellite classes (ATA, AATA) are frequently associated with an evolutionarily older subclass of Alu repeats, AluSx, whereas most of GATA and CA microsatellites are associated with a recent Alu subfamily, AluY. Our observations support all three possible mechanisms for the association of Alu repeats to microsatellites. Primers designed using a set of sequences from a particular microsatellite class showed higher homology with more sequences of that class than probes designed for other classes. We developed an efficient method of prescreening GGAA and ATA microsatellite clones for Alu repeats with probes designed in this study. We also showed that Alu probes labeled in a single reaction (multiplex labeling) could be used efficiently for prescreening of GGAA clones. Sequencing of these prescreened GGAA microsatellites revealed only 5% Alu repeats. Prescreening with primers designed for ATA microsatellite class resulted in the reduction of the loss of markers from approximately 50% to 10%. The new Alu probes that were designed have also proved to be useful in Alu-Alu fingerprinting.

Novel mutations and a mutational hotspot in the MODY3 gene.

Maturity-onset diabetes of the young 3 (MODY3) is a type of NIDDM caused by mutations in the transcription factor hepatocyte nuclear factor-1alpha (HNF-1alpha) located on chromosome 12q. We have identified four novel HNF-1alpha missense mutations in MODY3 families. In four additional and unrelated families, we observed an identical insertion mutation that had occurred in a polycytidine tract in exon 4. Among those families, one exhibited a de novo mutation at this location. We propose that instability of this sequence represents a general mutational mechanism in MODY3. We observed no HNF-1alpha mutations among 86 unrelated late-onset diabetic patients with relative insulin deficiency. Hence mutations in this gene appear to be most strongly associated with early-onset diabetes.

Identification and characterization of the mouse obesity gene tubby: a member of a novel gene family.

The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.

Chromosomal assignment of 2900 tri- and tetranucleotide repeat markers using NIGMS somatic cell hybrid panel 2.

Two thousand nine hundred and thirty-one tri- and tetranucleotide short tandem repeat polymorphisms (STRPs) developed by the Cooperative Human Linkage Center were assigned to chromosomes using the NIGMS somatic cell hybrid mapping panel 2 and an efficient pooling strategy. Approximately 82% of all STRPs tested were assigned by this method, with 96.7% accuracy. Many of the single chromosome cell lines contained portions of additional chromosomes, confirming previous reports. The cell lines for chromosomes 6, 14, and 20 contained extensive portions of other chromosomes. Five previously unreported chromosomal contaminants were identified and are reported. A new pooling strategy was designed to minimize ambiguous assignments.

Development of a screening set for new (CAG/CTG)n dynamic mutations.

The expansion of a (CAG/CTG)n triplet repeat has been found to be associated with at least seven genetic diseases, suggesting that this mechanism of disease may be fairly common. To accelerate the discovery of new loci containing (CAG/CTG)n triplet expansions, we have isolated numerous genomic clones containing this class of repeats. We have developed 338 sequence-tagged sites (STSs) containing (CAG/CTG)n repeat sequences. Two hundred ninety-nine STSs were unambiguously assigned to chromosomes, and 89 of the total were assigned to YACs. The 141 STSs that were developed based on (CAG/CTG)n repeats of at least seven units were genotyped on four reference CEPH individuals to estimate their polymorphic quality.

A small-insert bovine genomic library highly enriched for microsatellite repeat sequences.

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA.GT) > 12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of > 100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers.

Survey of trinucleotide repeats in the human genome: assessment of their utility as genetic markers.

Genetic markers based upon PCR amplification of short tandem repeat-containing sequence tagged sites (STSs) have become the standard for genetic mapping. We have completed a survey based on the direct isolation of representative members of each of the 10 trinucleotide repeat classes to determine their relative abundance, repeat size distribution, and general utility as genetic markers. Trinucleotide repeats, depending on the repeat class, are one to two orders of magnitude less frequent than (AC)n repeats. The average size of trinucleotide repeats sequenced was less than 15 repeat units in length, and only three of the STSs developed for this study demonstrated more than 25 repeats units. The (AAT)n class of repeats are the most abundant and also the most frequently polymorphic. Other classes of trinucleotide repeat classes observed to be frequently polymorphic include (AAC)n, (ACT)n, (ATC)n and (AAG)n; however, the relative abundance of these classes is less than that observed for the (AAT)n class of repeats. Based upon this initial survey, we have initiated saturation cloning of the (AAT)n class of repeats. At the time of submission of this manuscript, we have developed, as part of the Cooperative Human Linkage Center (CHLC), more than 415 new high heterozygosity (AAT)n genetic markers (more than two alleles in four individuals) and 200 new low heterozygosity (AAT)n STSs from this larger screening effort combined with the initial survey.

A collection of tri- and tetranucleotide repeat markers used to generate high quality, high resolution human genome-wide linkage maps.

We report a collection of tri- and tetranucleotide repeat sequence polymorphic markers used to construct genome-wide human linkage maps. Using a strategy of marker selection to create libraries highly enriched for the presence of specific tandem repeat elements, we have developed over 2000 high heterozygosity, easy-to-use tri- and tetranucleotide short tandem repeat polymorphisms (STRPs). To date, over 1300 of these markers have been genotyped on the CEPH reference families. Additional STRPs were assigned to chromosomes using human monochromosomal somatic cell hybrids. The linkage maps constructed with these markers have been integrated with other CEPH genotypes into a comprehensive high density linkage map. These STRPs have been shown to be robust for genotyping in a variety of laboratories using a variety of methods. The high quality of these STRPs makes them ideal candidates for use in genome-wide linkage searches. The integration of these markers with physical mapping reagents and other genetic markers will create a resource for moving from genome-wide linkage searches to rapid sublocalization of disease loci.

MATS: a rapid and efficient method for the development of microsatellite markers from YACs.

In this report, we describe the successful application of a rapid and efficient procedure, based on subtractive hybridization and PCR amplification, for generating microsatellite-based markers directly from yeast artificial chromosomes (YACs). This strategy, termed MATS (marker addition through subtraction), exploits the fact that the only difference between a yeast host strain harboring a YAC and the host strain alone is the artificial chromosome. Given the low complexity of the yeast genome and relatively large target size presented by a YAC, only a single round of subtraction is required before amplification of the target sequences (YAC) and cloning into a plasmid vector for further analysis. Several key steps have been designed to achieve optimal subtraction and to obtain preferential amplification and recovery of the target sequences. Methods for efficient construction of small insert libraries and rapid, nonradioactive screening have also been integrated into the protocol. Using a 750-kb YAC as a target, we identified a minimum of 14 unique microsatellite containing clones, leading to the development of 12 polymorphic STSs (sequence-tagged sites). These new markers will facilitate the genetic localization of targeted locus and allow the accurate ordering by STS content mapping of a cloned contig spanning the interval. In addition to the utility of this approach in positional cloning, this strategy may provide an approach for filling gaps in the emerging genetic maps.