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PubMed2008

Runs of homozygosity in European populations.

Estimating individual genome-wide autozygosity is important both in the identification of recessive disease variants via homozygosity mapping and in the investigation of the effects of genome-wide homozygosity on traits of biomedical importance. Approaches have tended to involve either single-point estimates or rather complex multipoint methods of inferring individual autozygosity, all on the basis of limited marker data. Now, with the availability of high-density genome scans, a multipoint, observational method of estimating individual autozygosity is possible. Using data from a 300,000 SNP panel in 2618 individuals from two isolated and two more-cosmopolitan populations of European origin, we explore the potential of estimating individual autozygosity from data on runs of homozygosity (ROHs). Termed F(roh), this is defined as the proportion of the autosomal genome in runs of homozygosity above a specified length. Mean F(roh) distinguishes clearly between subpopulations classified in terms of grandparental endogamy and population size. With the use of good pedigree data for one of the populations (Orkney), F(roh) was found to correlate strongly with the inbreeding coefficient estimated from pedigrees (r = 0.86). Using pedigrees to identify individuals with no shared maternal and paternal ancestors in five, and probably at least ten, generations, we show that ROHs measuring up to 4 Mb are common in demonstrably outbred individuals. Given the stochastic variation in ROH number, length, and location and the fact that ROHs are important whether ancient or recent in origin, approaches such as this will provide a more useful description of genomic autozygosity than has hitherto been possible.

Adolescent↗
PubMed2004

Identification and characterization of novel endogenous proteolytic forms of the human angiogenesis inhibitors restin and endostatin.

Restin and endostatin are C-terminal fragments of the noncollagenous domains of collagen XV and collagen XVIII exhibiting high sequence homology. Both polypeptides are distinguished by strong anti-angiogenic activity in vivo restricting the growth of solid tumors and metastasis. They are therefore currently being tested in clinical trials as anti-cancer drugs. We present the identification of new endogenous variants of both angiogenesis inhibitors isolated from a human hemofiltrate peptide library. Using an immunological screening approach with time-resolved rare earth metal fluorometry, immunoreactive compounds were purified chromatographically and characterized by mass spectrometry. We discovered four novel proteolytic products of restin as well as four variants of endostatin. Two endostatin products were characterized as short internal fragments (R176-L215 and R176-S219) of the entire molecule containing the recently identified beta1 integrin receptor binding site, which plays a major role in endothelial cell migration and angiogenesis. Two additional forms contain mucin-type O-glycosylations. The O-glycosylated variants possess an oligosaccharide unit consisting of one N-acetylgalactosamine (GalNAc), one N-acetylneuraminic acid (NANA) and two galactose residues (Gal) occurring as sialo-(V117-S311-GalNAc-Gal2-NANA) and asialoglycopeptides (V117-S311-GalNAc-Gal2). The four restin variants (R(I)-R(IV)) were identified with identical C- but different N-termini and no posttranslational modification (R(I): P66-A254, R(II): P75-A254, R(III): Y81-A254 and R(IV): A89-A254). Following a differential peptide mass fingerprint approach by reflector mode MALDI-TOFMS, the disulfide patterns of these circulating restins were determined as Cys1-Cys4 and Cys2-Cys3. These endogenous circulating collagen fragments will help to understand the physiological processing of the therapeutic proteins.

Amino Acid Sequence↗
PubMed2005

High-throughput assays for lipases and esterases.

In the past few years a considerable number of high-throughput screening (HTS) systems have been developed, especially for lipases and esterases. In this review, a range of HTS methods for the directed evolution of these hydrolases are covered. This includes spectrophotometric and fluorimetric formats as well as other approaches to allow for fast, efficient and reliable identification of desired enzyme variants within large mutant libraries. In addition, methods for library creation and application of lipases and esterases are briefly covered.

Directed Molecular Evolution↗
PubMed2004

Identification of two alphaRYR alleles and characterization of alphaRYR transcript variants in turkey skeletal muscle.

We hypothesized that a mutation in the turkey skeletal muscle ryanodine receptor alpha isoform (alphaRYR) underlies turkey meat quality problems which are strikingly similar to pale, soft, exudative (PSE) pork. RT-PCR analysis of turkey alphaRYR mRNA covering amino acids 376 to 615 (numbered according to the human sequence) revealed at least three transcript variants. One transcript was homologous to the mammalian skeletal muscle RYR1 sequence in this region. The second transcript variant (AS-81) was characterized by the absence of 81 bases located at the beginning of exon 13, while the third transcript variant (AS-193) carried a deletion of 193 bases, corresponding to the entire exon 13. Two alphaRYR genomic DNA alleles (alphaRYR-I and alphaRYR-II) carrying the region of deletions in the turkey cDNA sequences were identified. Nucleotide sequence analysis demonstrated that the two alleles are identical in exon sequences but different in intron sequences. Comparison of genomic and cDNA sequences indicated that both AS-81 and AS-193 transcript variants probably arose via alternative splicing. Consistent with this mechanism, the last eight nucleotides of the 81 bases form a consensus sequence for a splice acceptor site. Both alleles could give rise to the AS-81 and AS-193 transcript variants via alternative splicing. Birds homozygous for alphaRYR-II tended to have superior meat quality indicators including significantly higher muscle pH at 15-min post mortem and lower muscle exudate at 24-h post mortem, compared to birds homozygous for alphaRYR-I.

Alleles↗
PubMed2006

CRELD2: gene mapping, alternate splicing, and comparative genomic identification of the promoter region.

CRELD2 is the second member of the CRELD family of proteins. The only other CRELD family member, encoded by CRELD1, is also known as the AVSD2 gene as mutations in CRELD1 are associated with cardiac atrioventricular septal defects (AVSD). Like CRELD1, CRELD2 is ubiquitously expressed during development and by mature tissues. Recently, a specific CRELD2 isoform (CRELD2beta) was implicated as a regulator of alpha4beta2 nicotinic acetylcholine receptor expression, suggesting that the CRELD family has widely diverse biological roles in both developmental events and subsequent cell function. Here we report additional characterization of CRELD2, which was undertaken to further our understanding of this important family. Mapping of CRELD2 by FISH shows that it maps to 22q13 rather than the GenBank reported locus of 22p13. Comparative genomic analysis of upstream sequences shows a discrete region that is highly conserved among diverse species with hallmark features indicative of a promoter region. Functional analysis demonstrates that this region has promoter activity. Consistent with widespread expression of CRELD2, this region is GC-rich and lacks a TATA box. Overall, the highest levels of CRELD2 expression occur in adult endocrine tissues. However, alternative splicing of CRELD2 is extensive with positive identification of several splice variants expressed by most normal fetal and adult tissues. Confirmed splice variants encode 5 different CRELD2 isoforms that differ significantly in composition indicating that CRELD2 function is varied and as yet poorly understood.

Alternative Splicing↗
PubMed2025

Costs and cost-effectiveness of returning secondary findings from genomic sequencing based on the return of additional findings in the 100,000 Genomes Project.

PURPOSE: To assess costs and cost-effectiveness of returning additional findings from genome sequencing using data from the 100,000 Genomes Project (100kGP). METHODS: A model-based cost-utility analysis combining yield, consent rates, and cost data from the 100kGP with published estimates of downstream costs and quality-adjusted life years expected to accrue over a lifetime, after the identification of a pathogenic variant. RESULTS: The cost of returning additional findings to participants in the 100kGP was £7.1m or £81 per participant, with a yield of 0.85% for consented participants. The estimated lifetime incremental cost per participant was £125 and quality-adjusted life years 0.004, giving an incremental cost-effectiveness ratio of £28,830. Implementing a policy of returning additional findings is unlikely to be cost-effective (ie, 13%) at a willingness-to-pay threshold of £20,000. A short-term cost of returning findings of £43 per participant or lower (compared with the base case of £81) would result in an incremental cost-effectiveness ratio of less than £20,000. Alternatively, cost-effectiveness may be improved by returning additional findings to younger patient populations. CONCLUSION: Return of additional findings following genome sequencing for this group of conditions may not be a cost-effective use of health care system resources. Our cost-effectiveness outcomes rely on published estimates and should be validated through long-term follow-up data.

Humans↗
PubMed2006

Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides.

Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.

Amyloid beta-Peptides↗
PubMed2026

A novel missense mutation in tropomyosin 1 gene associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common genetic heart disorder that can lead to heart failure or sudden death. Family-based identification of rare sarcomeric variants can support molecular diagnosis and cascade screening in inherited HCM. This study aimed to identify and evaluate a novel TPM1 variant found in a Vietnamese family with HCM. The proband, a 3-year-old boy diagnosed with HCM, and eight relatives from three generations underwent clinical and genetic evaluation. A candidate variant initially identified by targeted next-generation sequencing was validated by PCR and Sanger sequencing. Familial segregation analysis was performed, and variant pathogenicity was assessed according to ACMG guidelines with support from in silico prediction and structural modeling. Sanger sequencing confirmed a heterozygous missense variant in exon 6 of TPM1 NM_001018005.2:c.576G > C, p.(Glu192Asp), in the proband, his father, and paternal grandfather, all of whom exhibited clinical signs of HCM. The variant was absent in unaffected relatives and in public population databases. Based on ACMG criteria (PM1, PM2, PM5, and PP3), the variant was classified as likely pathogenic. This novel TPM1 variant segregated with HCM in a Vietnamese family, expands the known mutational spectrum of TPM1 in hypertrophic cardiomyopathy, and warrants further functional investigation and familial genetic evaluation.

American College of Medical Genetics and Genomics ↗
PubMed2026

Genome-wide variation analysis of two Salvia hispanica L. genotypes and implication for associations with metabolic and adaptive traits.

BACKGROUND: Advances in next-generation sequencing have accelerated genome-wide exploration of genetic diversity in underutilized oilseed crops. Salvia hispanica L. (chia), a high-nutrient pseudocereal rich in omega-3 fatty acids, is increasingly valued for its health benefits and commercial potential, yet it remains poorly characterized at the genomic level. Understanding the scale and nature of genomic variation is essential for improving complex traits such as oil yield, stress tolerance, and seed quality. METHODS: Two contrasting chia genotypes, Black-chia (CACH-B) and White- chia (CACH-W), were resequenced using the Bio-Resequencing Toolkit (BRT) pipeline. High-coverage sequencing, with a mapping rate exceeding 99% and an average depth of approximately 28×, facilitated the detection and annotation of single-nucleotide polymorphisms (SNPs), insertions and deletions (InDels), copy-number variations (CNVs), and structural variants (SVs). The functional classification of variant impacts enabled the identification of genes potentially linked to metabolic and adaptive traits. RESULTS: A total of 1.97 million SNPs, 401,493 InDels, 836 CNVs, and 15,288 SVs were identified across the chia genome. Notably, approximately 53% of exonic SNPs were non-synonymous (dN/dS ≈ 1.28), predominantly affecting lipid metabolism, transcriptional regulation, and stress response pathways, potentially altering key agronomic traits. In addition, CNV hotspots were concentrated in chromosomes 3 and 6, overlapping MYB, WRKY, and bZIP transcription factor loci, may potentially be involved in stress tolerance and yield. Furthermore, structural rearrangements, including inversions and duplications within the FAD2, FAD3, and CYP450 gene clusters, were potentially associated with seed pigmentation and omega-3 biosynthesis, pointing to their potential breeding relevance. Observed heterozygosity (Hₒ ≈ 0.71) and nucleotide diversity (π ≈ 7 × 10-3) indicated moderate to high allelic richness. In addition, the low FST value (0.038) indicates substantial genomic similarity between the two genotypes. CONCLUSION: This study presents the first comprehensive map integrating SNPs, CNVs, and SVs in S. hispanica L. The results reveal a structurally dynamic genome characterized by substantial sequence and structural variation, providing valuable insights into genomic diversity and potential adaptive mechanisms in chia. The coexistence of high SNP diversity and abundant structural variation underpins chia's nutritional specialization and environmental resilience. These results deliver a foundational genomic resource for marker-assisted breeding, genome-wide association studies, and the development of climate-resilient chia cultivars.

Copy-number variation, structural variation↗
PubMed2005

Real-time RT-PCR analysis of two epitope regions encoded by the VP2 gene of infectious bursal disease viruses.

Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in chickens and leads to severe economic losses in the poultry industry. Vaccination may not be effective if there is exposure of the vaccinated flock to a different antigenic subtype, which reinforces the importance of identification of new IBDV variants. The virus outer capsid is constituted of VP2, in which the major neutralizing epitopes are located. Forty-eight bursa samples collected from IBDV infected commercial broiler flocks in the US were analyzed by real-time RT-PCR using probes designed for two epitope regions of VP2 denominated minor peak 1 and peak B. It was observed that 23, 48 and 44 samples tested with the minor peak probes Del-E, STC and F15, respectively, had a lower melting temperature (Tm) than expected. Furthermore, 44, 41 and 48 samples tested with the Del-E, STC and F15 peak B probes, respectively, had a lower Tm compared to the control, which indicates the presence of one or more nucleotide mutations in the samples. This fact was confirmed by nucleotide sequencing which also demonstrated that most mutations resulted in amino acid substitutions. Real-time RT-PCR can be a useful tool to assist in the development of more effective vaccination strategies.

Amino Acid Sequence↗
PubMed2004

Analysis of multiple Invs transcripts in mouse and MDCK cells.

Infantile nephronophthisis is associated with cystic kidneys, situs inversus, and INVS mutations. The function of the INVS product, inversin, is unknown but evidence suggests there are multiple inversin isoforms with differing molecular weights, cellular localization patterns, and binding partners. We used Northern blots, RT-PCR, and sequence analysis to identify alternative INVS transcripts. Northern blots probed with Invs cDNA detected four bands in normal mouse kidney. RT-PCR of mouse kidney RNA revealed Invs transcripts with skipping of exon 5, 11, or 13. We sequenced canine (MDCK-II cells) INVS and determined that the corresponding full-length protein shares identity with mouse (74%) and human (84%) inversin. Canine INVS produces a transcript that skips exon 12. Exon skips cause loss of inversin protein motifs, including ankyrin repeats, IQ domains, destruction boxes, and nuclear localization signals. Identification of INVS splice variants will help us determine which inversin protein motifs contribute to left-right asymmetry and kidney development.

Alternative Splicing↗
PubMed2005

Antibody humanization by framework shuffling.

We report here the humanization of a mouse monoclonal antibody (mAb B233) using a new technique which we call framework shuffling. mAb B233 was raised against the human receptor tyrosine kinase EphA2 which is selectively up-regulated in many cancer cell lines and as such constitutes an attractive target for cancer therapy. The six CDRs of B233 were fused in-frame to pools of corresponding individual human frameworks. These human frameworks encompassed all known heavy and light (kappa) chain human germline genes. The resulting Fab combinatorial libraries were then screened for binding to the antigen. A two-step selection process, in which the light and heavy chains of the parental mAb were successively humanized, resulted in the identification of several humanized variants that retained binding to EphA2. More precisely, after conversion to human IgG1, the dissociation constants of three select fully humanized variants ranged from 3 to 48 nM. This brings the best framework-shuffled, humanized binder within 5-fold of the avidity of parental mAb B233. Importantly, these humanized IgGs also possessed biochemical activities similar to those of parental mAb B233 as judged by induction of EphA2 phosphorylation. Thus, without requiring any rational design or structural information, this new humanization approach allows to rapidly identify various human framework combinations able to support the structural feature(s) of the CDRs which are essential for binding and functional activity.

Amino Acid Sequence↗
PubMed2002

Identification and recombinant production of human laminin alpha4 subunit splice variants.

Laminins, the major basement membrane glycoproteins, are composed of three subunits. We identified a splice variant of the human laminin alpha4 subunit transcript containing 21 extra nucleotides. A heptapeptide sequence, MDCPTIS, was inserted close to the two cysteine residues possibly involved in the intersubunit disulfide bonds. Both the authentic alpha4 subunit (alpha4A) and the variant with the heptapeptide insertion (alpha4B) were readily secreted as laminin-8 trimers (alpha4Abeta1gamma1 or alpha4Bbeta1gamma1) upon cotransfection with expression vectors for the beta1 and gamma1 subunits. The purified recombinant laminin-8 containing the alpha4B subunit was more potent in promoting cell spreading than that containing alpha4A, raising the possibility that the alternative splicing of the alpha4 subunit transcript regulates the cell-adhesive activity of laminin-8. Since both alpha4A and alpha4B transcripts were detected by RT-PCR in several human cell lines, these two isoforms of laminin-8 with differing cell-adhesive activities are present in the basement membranes of human tissues.

Alternative Splicing↗
PubMed1984

Fecal continence following an anterior-sagittal ano-enteroplasty in a patient with cloacal exstrophy.

A two-year-old Latin American female was referred for treatment of what appeared to be a straightforward case of cloacal exstrophy. At operation, incision along the superior margin of the exstrophic bladder allowed identification of an unusual variant of exstrophy which consisted of bladder exstrophy; erminal colon duplication, the common medial wall of which protruded 8 cm beyond the end of the bowel lumen, and which exited ventrally and immediately caudad to the exstrophic bladder; an enteric diverticulum at the superior rim of the bladder; and two small vaginae, existing laterally on either side, at the junction between the bladder and the terminal bowel. The terminal bowel was mobilized, the duplications were excised, and a tapering enteroplasty was performed. Despite a flat-appearing bottom, a posterior muscle complex was identified which extended anterolaterally and attached to the pubis on either side. Following observation of an appropriate response to electrical stimulation, the muscle complex was partially divided in the midline, from anterior to posterior, just enough to make room for the tapered bowel. The muscle was then reapproximated in the midline anteriorly, surrounding the bowel, and an anoplasty was constructed. The bladder was closed, and the pubic rami were wired together (bilateral posterior iliac osteotomies were performed at the beginning of the operation). One and 1/2 years following this procedure, the child has a normal "rectal" exam with a palpable sphincter. She has 3 to 4 solid bowel movements daily without soiling, and she awaits genitourinary reconstruction.

Abnormalities, Multiple↗
PubMed2002

The molecular epidemiology of Echinococcus granulosus and cystic hydatid disease.

It is now generally recognized that the dog tapeworm Echinococcus granulosus, the cause of cystic hydatid disease, exhibits substantial genetic diversity. This variability has important implications for the design and development of vaccines, diagnostic reagents and drugs effective against this parasite. The paper describes various deoxyribonucleic acid-based approaches that have been used for accurate identification of these genetic variants and their application in molecular epidemiological surveys of cystic hydatid disease in different geographical settings.

Animals↗
PubMed2000

5-HT7 receptors: current knowledge and future prospects.

Identification of three splice variants of the 5-HT7 receptor suggests a possible diversity in 5-HT7 receptor action. Indeed, 5-HT7 receptors have been implicated in the pathophysiology of several disorders; they play a role in smooth muscle relaxation within the vasculature and in the gastrointestinal tract. However, most of these assignments are derived from receptor localization studies and investigations using nonselective ligands, and are therefore mainly suggestive. The development of selective 5-HT7 receptor antagonists will be of utmost importance in determining the actual physiological and pharmacological roles of this receptor. Major challenges of 5-HT7 receptor research are determination of the transcriptional regulation of the gene encoding the 5-HT7 receptor and elucidation of the differences in regulation and signalling of its four gene products.

Alternative Splicing↗
PubMed2000

Alternative splicing of murine SorCS leads to two forms of the receptor that differ completely in their cytoplasmic tails.

We report the identification of a splice variant of SorCS, a member of the family of VPS10 domain containing receptors. These type I transmembrane proteins share the presence of internalization signals in their cytoplasmic tail as one common characteristic. We show that an alternatively spliced transcript of SorCS is generated by differential processing of a composite internal/terminal exon. This splice variant encodes a protein with an N-terminal VPS10 domain followed by a leucine-rich module and a transmembrane domain identical with the already described SorCS protein, but a divergent cytoplasmic tail. In contrast to the known intracellular regions of the related receptors, this splice variant contains no internalization or sorting signals.

Alternative Splicing↗
PubMed1997

Identification and comparison of point mutations associated in classic and variant infectious bursal disease viruses.

Restriction enzymes (RE) were used to identify point mutations in the nucleotide sequences of the infectious bursal disease virus (IBDV) strains Del-E, Del-A, STC, IN, Bursine 2, and Bio-Burs. The point mutations at amino acid sites 222, 254 and 323 were identified using REs BstNI, StyI and MboI, respectively. The nucleotide sequences of STC, IN, Bursine 2 and Bio-Burs were determined at each of these sites. Nucleotide sequence analysis using this data and previously reported data for Del-E and Del-A was used to confirm the point mutation and predict the resulting amino acids. Although there were some exceptions, the BstNI and StyI enzymes detected point mutations in the first base of the 222 and 254 codons, respectively and could be used to predict an amino acid change in the viruses. MboI was detecting a mutation in the third base of codon 323 and could not reliably predict an amino acid change at that position. The results indicated that amino acids 222 and 254 were consistently mutated in the variant viruses examined and that amino acid 323 was not. Furthermore, point mutations resulting in amino acid changes at position 222 suggested that several groups of viruses may be defined by this site alone; proline for classic strains like STC, threonine for Del-E and GLS, serine for IN, Bursine 2, and Bio-Burs and glutamine for Del-A.

Animals↗