Biallelic PI4KA variants cause neurological, intestinal and immunological disease.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα/PI4KA/OMIM:600286) is a lipid kinase generating phosphatidylinositol 4-phosphate (PI4P), a membrane phospholipid with critical roles in the physiology of multiple cell types. PI4KIIIα's role in PI4P generation requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions. Here we show that biallelic PI4KA sequence alterations in humans are associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, affected individuals may present with inflammatory bowel disease, multiple intestinal atresia and combined immunodeficiency. Our cellular, biochemical and structural modelling studies indicate that PI4KA-associated phenotypical outcomes probably stem from impairment of PI4KIIIα-TTC7-FAM126's organ-specific functions, due to defective catalytic activity or altered intra-complex functional interactions. Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insight into the combinatorial biology of the PI4KIIIα-FAM126-TTC7-EFR3 molecular complex.

Promoting Choosing Wisely Thyroid Function Test Guidelines in a Large Pediatric Hospital System.

BACKGROUND: Free thyroxine (fT4) is often ordered when not indicated. The goal of the current study was to use quality improvement tools to identify and implement an optimal approach to reduce inappropriate fT4 testing throughout a large pediatric hospital system. METHODS: After reviewing evidence-based guidelines and best practices, a thyroid-stimulating hormone with reflex to fT4 test and an outpatient thyroid order panel with clinical decision support at order entry, along with several rounds of provider education and feedback, were implemented. Outpatient and inpatient order sets and system preference lists were reviewed with subject matter experts and revised when appropriate. Tracking metrics were identified. Automated monthly run charts and statistical process control charts were created using data retrieved from the electronic health record. Charts established baseline data, balancing measure data, monitored the impact of interventions, and identified future interventions. RESULTS: Over a 44-month period, among nonendocrinology providers, a reduction in fT4 and thyroid-stimulating hormone co-orders from 67% to 15% and an increase in reflex fT4 tests from 0% to 77% was obtained in inpatient and outpatient settings. Direct cost savings as a result of performing 5179 fewer fT4 tests over 3 years was determined to be $45 800. CONCLUSIONS: After implementation of a reflex fT4 test, a novel order panel with clinical decision support, provider education, and changes to ordering modes, a large and sustainable reduction in fT4 tests that was associated with significant cost savings was achieved among nonendocrinology providers.

Thyrotropin receptor/thyroglobulin messenger ribonucleic acid in peripheral blood and fine-needle aspiration cytology: diagnostic synergy for detecting thyroid cancer.

RT-PCR for thyroglobulin (Tg) and TSH receptor (TSHR) mRNA has been used to detect circulating thyroid cancer cells. Little is known, however, regarding the preoperative sensitivity of this test to detect cancer. Seventy-two patients with thyroid disease (36 with malignancy and 36 with benign disease) were evaluated preoperatively. TSHR and Tg mRNA transcripts were detected by RT-PCR assays, previously determined to be specific for cancer cells. There was 100% concordance between TSHR and Tg mRNA RT-PCR results. Of 36 cancer patients, 11 had recurrent disease, and all were positive by RT-PCR. Among 25 patients with no prior thyroid surgery, 18 tested positive preoperatively (sensitivity 72%). Seven of 36 patients with benign disease tested positive (specificity 80%). The overall preoperative diagnostic accuracy was 77%. Preoperative fine-needle aspiration (FNA) biopsy was performed on 46 of 61 patients with no prior thyroid surgery. FNA was diagnostic in 28 (61%) patients. Preoperative cytology was adequate but not diagnostic in 18 (39%) patients. RT-PCR correctly classified 14 of these 18 patients with indeterminate FNA, and the test detected three of four cancer patients as positive (75% sensitive) and 11 of 14 patients (78% specific) with benign disease as negative. The combined diagnostic performance characteristics for RT-PCR and FNA cytology were sensitivity = 95%, specificity = 83%, and diagnostic accuracy = 89%, with positive and negative predictive values of 84 and 95%, respectively. Our results suggest that the molecular detection of circulating thyroid cancer cells by RT-PCR for TSHR/Tg mRNA complements FNA cytology in the preoperative differentiation of benign from malignant thyroid disease and their combined use may save unnecessary surgeries.

Creation of a large-scale genetic data bank for cardiovascular association studies.

BACKGROUND: A prerequisite for undertaking genetic association studies is the need for a genetic data bank with adequate DNA samples and a well-described clinical cohort. METHODS: We initiated a prospective single-center study enrolling 6,273 patients referred for cardiac catheterization in a genetic data bank (with eventual goal of 10,000 enrollees). Using a prescreening tool, the patients had comprehensive clinical phenotyping, including angiogram, electrocardiogram, echocardiogram, clinical history, and medication profile (Appendix A). Along with this clinical information, DNA, serum, plasma, basic metabolic panel, inflammation, and lipid panel were collected and stored in the database. RESULTS: Mean age of the patients enrolled was 64 +/- 12 years; 69% are men, 26% have diabetes, 79% have dyslipidemia, and 72% have coronary artery disease (CAD) > or = 50%. We undertook extensive quality-control measures to ensure the validity of both the clinical and DNA samples acquired into our GenBank. As part of this validation, we undertook a genetic association study to discern the effect of the apoE4 polymorphism on the risk for atherosclerosis. We are able to show that the apoE4 polymorphism is an independent risk factor for CAD. CONCLUSIONS: We have been able to create a large-scale genetic data bank as a resource to undertake genetic association studies. Key elements in implementation of this GenBank and baseline characteristics of our patient cohort are summarized. Lastly, as a "proof of concept" for the utility of this resource to discern gene variants associated with disease, we validated apoE4 polymorphism as an independent risk factor for CAD.

A rare mutation in the primer binding region of the amelogenin gene can interfere with gender identification.

PCR amplification of part of the X-Y homologous amelogenin gene with a single primer pair has been used as a sex identification test because it generates different length products from the X and Y chromosomes. Using a commercially available kit that contains amelogenin primers, we report a single phenotypically normal Caucasian male out of 327 males tested to date that failed to show an X chromosome-specific PCR product. Using alternative amelogenin primers external to but encompassing the initial amplicon, an X chromosome-specific product was seen. Sequence analysis of this X-specific PCR product revealed a C to G mutation at the most 3' base of the initial reverse amelogenin PCR primer. An alternative reverse PCR primer with this most 3' base deleted showed X- and Y-specific products from the case study male. Rare mutations that result in a failure to amplify sex chromosome-specific products can result in incorrect gender identification.

Primary adenocarcinoma in a donor lung: evaluation and surgical management.

We report a case of primary non-small cell cancer diagnosed in the donor lung 33 months after bilateral pulmonary transplantation. The tumor was treated surgically. A lingular sparing left upper lobe bisegmental lung resection was performed.

A comparison of multiplex and monoplex T-cell receptor gamma PCR.

T-cell receptor gamma (TCRgamma) PCR is often used to detect clonal T-cell populations. Because TCRgamma contains a limited number of variable (Vgamma) and joining (Jgamma) regions, a small number of PCR primers can be used to assess T-cell clonality. The seven primers used in the current study were described previously and were split into 2 or 3 multiplex primer sets. In this study, a single 7-primer multiplex (7-plex) PCR reaction was compared with all 12 possible monoplex primer combinations on 18 samples previously analyzed for T-cell receptor rearrangements by TCRbeta Southern blot and/or TCRgamma PCR followed by temporal temperature gradient gel electrophoresis. Using fluorescent Vgamma-region primers, unlabeled Jgamma-region primers, and capillary electrophoresis, we show all TCRgamma rearrangements seen by 7-plex PCR on known positive samples were seen following monoplex PCR. However, additional TCRgamma gene rearrangements were seen in monoplex PCR reactions that were not seen in the 7-plex PCR reactions. Monoplex but not 7-plex PCR of known negative samples occasionally showed TCRgamma gene rearrangements, often with less frequently used Vgamma and Jgamma-region primers, and may have represented false positive results. In summary, the single 7-plex PCR reaction correctly identified specimens with TCRgamma clonal populations and represents an improvement over existing assays that use these same primers split into several smaller multiplex reactions. Monoplex PCR has no advantage over multiplex PCR and has the potential to lead to false positive results.

Detection of a novel point mutation of the prothrombin gene at position 20209.

Detection of the prothrombin G20210A mutation was performed on the LightCycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany) using commercially available primers and hybridization probes. Herein we report four cases from unrelated African American individuals where LightCycler analysis showed atypical melting curves. Sequence analysis of the four samples showed heterozygosity for a C to T mutation 1 bp upstream from the known 20210 mutation at position 20209. The clinical significance of this mutation is not known, but three patients in whom it was detected had a history of venous thrombosis or stroke. The fourth patient had severe liver disease, which may have masked thrombotic predisposition. Since most assays used to detect the G20210A mutation use a PCR/restriction digestion assay, which would not detect the C20209T mutation, this new mutation may be underrecognized when prothrombin gene mutation testing is performed by PCR/restriction digestion assay.

Locked nucleic acid probes for enhanced detection of FLT3 D835/I836, JAK2 V617F and NPM1 mutations.

AIMS: Detecting low-level clinically significant cancer-relevant somatic mutations can be difficult. Several technologies exist for detecting minority mutations. One method is locked nucleic acid (LNA) PCR. In this study, LNA probes were used to enhance the sensitivity for detecting FLT3 D835/I836 tyrosine kinase domain (TKD) mutations, the JAK2 V617F mutation and insertion mutations in the nucleophosmin 1 gene. METHODS: PCR was performed with and without LNA probes using DNA known to contain FLT3 D835/I836 TKD, JAK2 V617F and NPM1 mutations. FLT3 D835/I836 TKD mutations were detected following EcoRV restriction enzyme digestion and capillary electrophoresis. The JAK2 V617F mutation was detected by melt-curve analysis. NPM1 insertions were detected by capillary electrophoresis. RESULTS: The detection of FLT3 D835/I836, JAK2 V617F and NPM1 mutations was enhanced approximately 10-50-fold using LNA probes. Rare JAK2 double mutants gave abnormal blocking patterns with the LNA probe. CONCLUSIONS: Adding LNA probes to existing assays is a simple way to enhance and confirm the detection of mutations, especially those at low levels.

Multiplex ligation-dependent probe amplification for rapid detection of proteolipid protein 1 gene duplications and deletions in affected males and carrier females with Pelizaeus-Merzbacher disease.

BACKGROUND: Pelizaeus-Merzbacher disease is a rare X-linked neurodegenerative disorder caused by sequence variations in the proteolipid protein 1 gene (PLP1). PLP1 gene duplications account for approximately 50%-75% of cases and point variations for approximately 15%-20% of cases; deletions and insertions occur infrequently. We used multiplex ligation-dependent probe amplification (MLPA) to detect PLP1 gene alterations, especially gene duplications and deletions. METHODS: We performed MLPA on 102 samples from individuals with diverse PLP1 gene abnormalities and from controls, including 50 samples previously characterized for the PLP1 gene by quantitative PCR but which were anonymized for prior results and sex. RESULTS: All males with PLP1 gene duplications (n = 13), 1 male with a triplication, 2 males with whole gene deletions, and all controls (n = 72) were unambiguously assigned to their correct genotype. Of 4 female carriers tested by MLPA and quantitative PCR, 3 were duplication carriers by both methods, and 1 was a triplication carrier by MLPA and a duplication carrier by quantitative PCR. For 1 sample with a partial deletion, MLPA showed exon 3 deleted but PCR showed exons 3 and 4 deleted. Sequence analysis of 2 samples with reduced dosage for exons 3 and 5 revealed point variations overlapping the annealing site for the corresponding MLPA probe. The precision of MLPA analysis was excellent and comparable to or better than quantitative PCR, with CVs of 4.3%-9.8%. CONCLUSIONS: MLPA is a rapid and reliable method to determine PLP1 gene copies. Samples with partial PLP1 gene dosage alterations require confirmation with a non-MLPA method.

Primary progressive multiple sclerosis as a phenotype of a PLP1 gene mutation.

We report a 49-year-old woman with a history of progressive gait disturbance, white matter disease, and cerebrospinal fluid immunoglobulin abnormalities who met criteria for primary progressive multiple sclerosis and whose son died at age 10 years of an unknown congenital neurodevelopmental disorder. Sequencing of the proteolipid protein 1 gene showed a novel mutation, Leu30Arg (c.89TG), in the mother and son. Pelizaeus-Merzbacher disease is the cause of death in the son and explains the mother's adult-onset neurological disorder. This case goes against dogma that mothers of severely affected sons are asymptomatic as adults and expands the differential diagnosis of primary progressive multiple sclerosis to include proteolipid protein 1 gene mutations.

Pathologic clonal cytotoxic T-cell responses: nonrandom nature of the T-cell-receptor restriction in large granular lymphocyte leukemia.

T-cell large granular lymphocyte (T-LGL) leukemia is a clonal lymphoproliferation of cytotoxic T cells (CTLs) associated with cytopenias. T-LGL proliferation seems to be triggered/sustained by antigenic drive; it is likely that hematopoietic progenitors are the targets in this process. The antigen-specific portion of the T-cell receptor (TCR), the variable beta (VB)-chain complementarity-determining region 3 (CDR3), can serve as a molecular signature (clonotype) of a T-cell clone. We hypothesized that clonal CTL proliferation develops not randomly but in the context of an autoimmune response. We identified the clonotypic sequence of T-LGL clones in 60 patients, including 56 with known T-LGL and 4 with unspecified neutropenia. Our method also allowed for the measurement of clonal frequencies; a decrease in or loss of the pathogenic clonotype and restoration of the TCR repertoire was found after hematologic remission. We identified 2 patients with identical immunodominant CDR3 sequence. Moreover, we found similarity between multiple immunodominant clonotypes and codominant as well as a nonexpanded, "supporting" clonotypes. The data suggest a nonrandom clonal selection in T-LGL, possibly driven by a common antigen. In contrast, the physiologic clonal CTL repertoire is highly diverse and we were not able to detect any significant clonal sharing in 26 healthy controls.

Comparison of five methods for extraction of Legionella pneumophila from respiratory specimens.

The efficiencies of five commercially available nucleic acid extraction methods were evaluated for the recovery of a standardized inoculum of Legionella pneumophila in respiratory specimens (sputum and bronchoalveolar lavage [BAL] specimens). The concentrations of Legionella DNA recovered from sputa with the automated MagNA Pure (526,200 CFU/ml) and NucliSens (171,800 CFU/ml) extractors were greater than those recovered with the manual methods (i.e., Roche High Pure kit [133,900 CFU/ml], QIAamp DNA Mini kit [46,380 CFU/ml], and ViralXpress kit [13,635 CFU/ml]). The rank order was the same for extracts from BAL specimens, except that for this specimen type the QIAamp DNA Mini kit recovered more than the Roche High Pure kit.