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Small-colony variants of Staphylococcus aureus may cause invasive disease in adults that is prolonged and refractory to standard therapies. We present a case of sternoclavicular arthritis with small-colony variant S. aureus that occurred in an 11-year-old child and discuss the importance of identification of these variants in the clinical microbiology laboratory.
We previously isolated a complete set of primary trisomics along with many other aneuploids from triploid plants derived from an indica rice variety "Zhongxian 3037." About 30,000 progeny from these trisomic and aneuploid plants were grown each year from 1994 to 1999. The variants that differed morphologically from both the diploids and the original primary trisomics were collected for cytological identification. From these variants, a complete set of telotrisomics covering all 24 rice chromosome arms was obtained. The identities of the extra chromosomes were further confirmed by dosage analysis of the RFLP markers on extra chromosome arms. The telocentric nature of the extra chromosomes in these stocks was verified by fluorescence in situ hybridization (FISH) using a rice centromeric BAC clone as a marker probe. In general, the shorter the extra chromosome arm of a telotrisomic, the stronger the resemblance it bears to the diploid; the longer the extra chromosome arm, the stronger the resemblance to the corresponding primary trisomic. We demonstrated that DNA clones can be rapidly assigned to specific chromosome arms by dosage analysis with the telotrisomics. We also showed that telotrisomics are valuable tools for chromosome microdissection and for developing chromosome-specific DNA markers.
Transthyretin (TTR) is a 127-amino acid residue transport protein. In plasma, TTR exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR are hypothesized to destabilize the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with extracellular deposition of wild-type TTR, its variants or fragments as amyloid fibrils in various tissues and organs. A definitive diagnosis of ATTR depends on the detection and identification of TTR variants. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), in combination with trypsin digestion, have been shown to be powerful tools in characterizing TTR variants. Typically, TTR or its tryptic digest is analyzed by MALDI-TOF MS, liquid chromatography ESI MS, or both. Analysis of tryptic digests by MALDI-TOF MS does not provide enough sequence coverage in TTR to identify all possible modifications. To improve sequence coverage, aliquots of immunoprecipitated TTR samples were digested with trypsin, lysyl endopeptidase Lys-C, or endoproteinase Asp-N. Identification of the peptides from each digest by MALDI-TOF MS provided preliminary information about the sites and mass shifts due to amino acid substitutions from genetic mutations and to posttranslational modifications. The location and identity of the modifications in the variant proteins were then confirmed by tandem mass spectrometry, accurate mass measurements, and direct DNA sequence analysis. Using these methodologies, we achieved 100% sequence coverage. The detection of two nonpathologic variants (Thr119Met and Gly6Ser) and four pathologic variants (Phe64Leu, Asp38Ala, Phe44Ser, and previously unreported Trp41Leu) are described as illustrations of this approach.
A reversed-phase high-performance liquid chromatographic method for the separation of the most common and some less common genetic variants of the bovine caseins is described. When the method is used for analysing clarified skim milk, simultaneous identification of casein variants and major they protein variants can be effected in a single run. The potential of the method for quantitative application is discussed.
Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function comprises mainly subtypes 2A, 2B and 2M. The diagnosis of type 2 von Willebrand disease may be guided by the observation of a disproportionately low level of ristocetin cofactor activity or collagen-binding activity relative to the von Willebrand factor antigen level. The decreased platelet-dependent function is often associated with an absence of high molecular weight multimers (types 2A and 2B), but the high molecular weight multimers may also be present (type 2M and some type 2B), and supranormal multimers may exist (as in the Vicenza variant). Today, the identification of mutations in particular domains of the pre-provon Willebrand factor is helpful to classify these variants and to provide further insight into the structure-function relationship and the biosynthesis of von Willebrand factor. Thus, mutations in the D2 domain, involved in the multimerization process, are found in patients with type 2A, formerly named IIC von Willebrand disease. Mutations in the D3 domain characterize the Vicenza variant, or type IIE patients. Mutations in the A1 domain may modify the binding of von Willebrand factor multimers to platelets, either increasing (type 2B) or decreasing (types 2M and 2A/2M) the affinity of von Willebrand factor for platelets. In type 2A disease, molecular abnormalities identified in the A2 domain, which contains a specific proteolytic site, are associated with alterations in folding that impair the secretion of von Willebrand factor or increase its susceptibility to proteolysis. Finally, a mutation localized in the C terminus cysteine knot domain, which is crucial for the dimerization of von Willebrand factor subunit, has been identified in a rare subtype 2A, formerly named IID.
High performance liquid chromatography (HPLC) demonstrated advantages over conventional procedures employed in newborn and adult hemoglobinopathy screening programs for the identification of Hb variants has promoted the need to reassess our knowledge of hemoglobin reference ranges as it relates to HPLC quantitation. In this study, the HPLC hemoglobin reference ranges derived from 200 normal African American adults are expressed as follows: Hb A mean 93.6 percent (s.d. 1.3, ranges 89.8 to 95.2), Hb A1 mean 2.0 percent (s.d. 0.6, ranges 0.8 to 5.2), Hb F mean 3.2 percent (s.d. 0.7, ranges 1.7 to 5.3) and Hb A2 mean 1.2 percent (s.d. 0.4, ranges 0.5 to 3.4); while the HPLC results for normal newborns and babies (n = 99) in the African American population fluctuates from Hb F mean 82.0 percent (s.d. 7.7, range 66.6 to 89.9) and Hb A mean 19.0 percent (s.d. 7.7, ranges 10.1 to 33.4) at 4 days to a mean of 15. percent (s.d. 4.8, range 9.3 to 22.8) for Hb F and a mean of 85.0 percent (s.d. 5.1, ranges 76.4 to 90.7) for Hb A at 300 days after birth. In case of the most common hemoglobin variants for this population, it has been shown that the A/S and A/C ratios for adults (Hb AS, Hb AC) and newborns (Hb FAS, and FAC) remained within the 1.5 (range 1.0 to 2.2) limits regardless of age group. Application of these HPLC ranges to confront other abnormalities will prove most useful during blood screening processes.
CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C9*1, CYP2C9*2, CYP2C9*3, and CYP2C9*4. In the present study, DNA from 140 European Americans and 120 African Americans was examined by single-strand conformational polymorphism and restriction fragment length polymorphism analyses, resulting in the identification of a new CYP2C9 variant, CYP2C9*5. This variant is derived from a C1080G transversion in exon 7 of CYP2C9 that leads to an Asp360Glu substitution in the encoded protein. The CYP2C9*5 variant was found to be expressed only in African Americans, such that approximately 3% of this population carries the CYP2C9*5 allele. The variant was expressed in, and purified from, insect cells infected with a recombinant baculovirus. Comparative kinetic studies using the purified wild-type protein CYP2C9*1; the Ile359Leu variant, CYP2C9*3; and the Asp360Glu variant, CYP2C9*5 were carried out using (S)-warfarin, diclofenac, and lauric acid as substrates. The major effect of the Asp360Glu mutation was to increase the K(m) value relative to that of CYP2C9*1 for all three substrates: 12-fold higher for (S)-warfarin 7-hydroxylation, 5-fold higher for the 4'-hydroxylation of diclofenac, and 3-fold higher for the omega-1 hydroxylation of lauric acid. V(max) values differed less than K(m) values between the CYP2C9*1 and CYP2C9*5 proteins. In vitro intrinsic clearances for CYP2C9*5, calculated as the ratio of V(max)/K(m), ranged from 8 to 18% of CYP2C9*1 values. The corresponding ratio for CYP2C9*3 was 4 to 13%. Accordingly, the in vitro data suggest that carriers of the CYP2C9*5 allele would eliminate CYP2C9 substrates at slower rates relative to persons expressing the wild-type protein.
On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is demonstrated for high-resolution analysis of model proteins, human hemoglobin variants, and Escherichia coli proteins. The acquisition of high-resolution mass spectra of hemoglobin beta chains allows direct identification of hemoglobin variants A and C, differing in molecular mass by 1 Da. Direct mass determination of cellular proteins separated in the CIEF capillary is achieved using their isotopic envelopes obtained from ESI-FTICR. The factors which dictate overall performance of CIEF-ESI-FTICR, including duty cycle, mass resolution, scan rate, and sensitivity, are discussed in the context of protein variants and cell lysates analyzed in this study.
Thrombotic predisposition may affect pregnancy outcome, but in non-Caucasians the contributing genetic factors are poorly characterized. Two recently identified prothrombin gene mutations (20209C>T and 20221C>T) have been observed in non-Caucasian patients with thrombosis. The mutations are located near the commonly identified variant 20210G>A and have not been reported in Caucasian patients. The authors report a novel connection with pregnancy complications. The identification of sequence variants other than 20210G>A in the 3'-untranslated region of the prothrombin gene suggests that additional nucleotide substitutions may contribute to the development of thrombotic events and adverse pregnancy outcomes, especially in less well-characterized populations.
A closed low pressure liquid chromatography system (LPLC) is described which is suitable for haemoglobinopathy screening. The system (Glycomat) was originally developed for the quantitation of the glycated haemoglobin (HbA1). The same biochemical principles have been applied to the separation of haemoglobin A2 (HbA2) and haemoglobin variants. The instrument offers three modes of use including a fast haemoglobin elution, a variant screen and a HbA2 assay for thalassaemia screening. The fast screen isolates all of the common haemoglobin variants except HbE which elutes with HbA. This mode is a more rapid alternative to the Sickledex test. The variant screen produces a wider separation of abnormal variants giving an identification and quantitation for each. The HbA2 assay separates this minor fraction from all other haemoglobins giving an accurate percentage. Abnormal variants are also separated. To validate the HbA2 assay 252 samples were assayed by the cellulose acetate electrophoresis/elution method and LPLC with a correlation of 0.932. The system provides an accurate and sensitive alternative to traditional manual chromatography and electrophoresis methods. The automated sampler allows batches from 1-99 samples to be processed with significant savings in operator time.
MOTIVATION: The accurate and sensitive identification of de novo variants, which are unique to an individual and not found in the parents' germlines, is critical for understanding the genetic basis of rare diseases, developmental disorders, and evolutionary processes. Existing de novo variant detection pipelines often lack the flexibility to handle multiple variant types, struggle with speed and reproducibility across computational environments, demand extensive manual configuration, or require bioinformatics expertise for downstream curation and analysis, limiting their scalability and usability for large genomic studies. Accordingly, there is a pressing need to better address these challenges. RESULTS: We introduce TriosCompass, an open-source Snakemake workflow that addresses these challenges by providing a modular, accelerated, and environmentally-configurable end-to-end solution for comprehensive de novo variant discovery. It integrates state-of-the-art tools into a reproducible framework, empowering researchers to discover novel genetic insights with greater efficiency and reliability. AVAILABILITY: TriosCompass is implemented as a Snakemake workflow and is freely available at https://github.com/NCI-CGR/TriosCompass_v2 or on Zenodo (10.5281/zenodo.17981062). SUPPLEMENTARY INFORMATION: Supplementary data is available on GitHub at https://github.com/NCI-CGR/TriosCompass_v2/tree/manuscript/report_dashboards. Supplementary methods on DeepTrio benchmark runs can be viewed at: https://github.com/NCI-CGR/TriosCompass_v2/blob/manuscript/TriosCompass_Supp_Methods_deeptrio_benchmark.md.
The Bax gene is a member of the Bcl2 family that functions to regulate the programmed cell death process. A number of Bax isoforms have been previously identified: alpha, beta, gamma, delta, and omega. Here we report the identification and characterization of an additional Bax variant, termed Baxepsilon. The newly identified Bax variant contains a 97-base insertion generated by alternative splicing which includes a previously unidentified exon between exons 4 and 5. The insertion causes the production of a truncated Bax protein, termed Baxepsilon, which encodes a protein of 164 residues with a calculated molecular weight of 18 kDa. The last 69 amino acids of Baxalpha that encompass the BH2 and the TM domains are missing in Baxepsilon. The Baxepsilon protein, when expressed as a GST fusion protein, associated efficiently with Baxalpha, Baxepsilon, Bcl2, and Bcl-xL. In addition, Baxepsilon was active in inducing apoptosis when tested in a transient transfection assay. Furthermore, the presence of antiapoptotic genes including Bcl2, Bcl-xL, and baculovirus p35 abrogated Baxepsilon and Baxalpha function. Although the newly identified Bax variant was detectable by RT-PCR in several normal mouse tissues, the role of this variant in controlling programmed cell death is currently unknown.
Two splice variants of the 5-HT7 receptor were identified in human brain that differ in the lengths of their intracellular carboxy terminal tail. Identification of the variants of this receptor is of particular interest since the 5-HT7 receptor is known to have a high affinity for a number of antidepressants and is localized in brain regions thought to be implicated in depression. The two isoforms are expressed in roughly equal amounts in various regions of the human brain. When expressed in NIH-3T3 cells, both variants encode functional 5-HT7 receptors, positively coupled to adenylyl cyclase. We suggest that both variants are derived from a single gene by alternative mRNA splicing. Furthermore, our results from Southern blot analysis studies suggest that additional 5-HT7 receptor genes may exist in human.
BACKGROUND: Neurodevelopmental disorders are one of the most prevalent reasons for genetic testing in childhood. Despite the identification of over 1950 associated genes, many proposed candidate genes lack convincing gene-disease validity. The gene PREP encodes the broadly expressed prolyl endopeptidase whose exact function remains largely unknown. A homozygous PREP variant has been reported once as a candidate gene in two siblings with intellectual disability but no functional studies were conducted. METHODS: Exome and trio genome sequencing were performed in two unrelated families as part of larger cohorts. Segregation analysis, RNA sequencing and immunoblots were performed to further examine the pathogenicity of detected PREP variants. RESULTS: We report three individuals from two unrelated families who presented with intellectual disability, behavioural abnormalities, strabismus, generalised muscular hypotonia, dysmorphic facial features and epilepsy. Exome and genome sequencing identified two different homozygous rare PREP variants: c.1570_1573dup, p.(Asn525Thrfs*5) and c.1839-2A>G, p.?. RNA sequencing confirmed the detected intronic variant to result in two aberrant mRNA isoforms. In patient-derived cells immunoblots showed absence of PREP protein. CONCLUSION: Our data suggest PREP deficiency as the underlying cause of a syndromic neurodevelopmental disorder.
Acute myeloid leukemia (AML) with the t(8;21) (q22;q22) creating the AML1-ETO fusion gene is a distinct type of AML generally associated with a favorable prognosis. The clinicopathologic features of AML carrying variant t(8;21) are less well characterized. We report 4 cases of AML characterized by ins(8;21)(q22;q22q22), t(1;21;8)(q25;q22;q22), t(8;11;21)(q22;q13;q22), and t(4;21;8;12)(q31.3;q22;q22;q15), respectively. Fluorescence in situ hybridization or reverse transcriptase-polymerase chain reaction assay confirmed the presence of AML1-ETO or its transcripts in 3 cases assessed. Each neoplasm had morphologic characteristics of AML associated with the t(8;21). The blasts in 2 cases expressed CD19. All patients were treated with combination chemotherapy and are in complete remission, despite 2 relapses in 1 patient, with a follow-up period ranging from 8 to 46 months. We conclude that cases of AML with variant t(8;21) display morphologic, immunophenotypic, and clinical features similar to classical cases. A combination of conventional cytogenetic, and molecular analyses allows identification of these variants.
Using screening, electrophoretic and spectrophotometric examinations, the authors studied the polymorphism of serum cholinesterase (CE) (CP 3.1.1.8) in children with undifferentiated oligophrenia. The rates of the identified phenotypes were as follows: CHE1U--0.769; CHE1UD--0.039; CHE1UF--0.192; CHE2(5-)-1.000. The incidence of the CHE1UF variant of the enzyme considerably exceeded that in the control groups (u = 2.44; u = 3.23) while the frequency of the CHE1U variant detection was significantly below that in the control groups (u = 2.70; u = 3.01; u = 3.46). The author discusses the advisability of the identification of CE variants in patients with psychoneurological diseases in order to prevent the development of side effects of myorelaxants and local anesthetics.
Identification of factors that cause susceptibility to, and clinical expression of, variant Creutzfeldt-Jakob disease (vCJD) is essential for future management of the disease. We established MHC genotypes of 76 individuals with vCJD and 131 controls, and analysed MHC phenotypes in relation to age of onset of vCJD and its duration from presentation to death. There were no significant differences between vCJD and control populations in frequencies of any MHC types, nor were there associations between MHC type and age of onset or duration of vCJD disease. Our results do not support the idea of an association between MHC types and either susceptibility to, or expression of, vCJD.
BACKGROUND: Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. RESULTS: We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. CONCLUSION: Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility.