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DNA junctions (DNAJs) frequently impact clinically relevant genes in tumors and are important for diagnostic and therapeutic purposes. Although routinely screened through fluorescence in situ hybridization assays, such testing only allows the interrogation of single-gene regions or known fusion partners. Comprehensive assessment of DNAJs present across the entire genome can only be determined from whole-genome sequencing. Structural variance analysis from whole-genome paired-end sequencing data is, however, frequently restricted to copy number changes without DNAJ detection. Through optimized whole-genome sequencing and specialized bioinformatics algorithms, complete structural variance analysis is reported, including DNAJs, from formalin-fixed DNA. Selective library assembly from larger fragments (500 bp) and economical sequencing depths (300 to 400 million reads) provide representative genomic coverage profiles and increased allelic coverage to levels compatible with DNAJ calling (40× to 60×). Although consistently fragmented, more recently formalin-fixed, specimens (2 years' storage) revealed consistent populations of larger DNA fragments. Optimized bioinformatics efficiently detected90% of DNAJs in two prostate tumors (approximately 60% tumor) previously analyzed by mate-pair sequencing on fresh frozen tissue, with evidence of at least one spanning-read in 99% of DNAJs. Rigorous masking with data from unrelated formalin-fixed tissue progressively eliminated many false-positive DNAJs, without loss of true positives, resulting in low numbers of false-positive passing current filters. This methodology enables more comprehensive clinical genomics testing on formalin-fixed clinical specimens.

Background Genomic aberrations have been identified in metastatic castration-resistant prostate cancer (mCRPC), but molecular predictors of resistance to abiraterone acetate/prednisone (AA/P) treatment are not known. Patients and methods In a prospective clinical trial, mCRPC patients underwent whole-exome sequencing (n = 82) and RNA sequencing (n = 75) of metastatic biopsies before initiating AA/P with the objective of identifying genomic alterations associated with resistance to AA/P. Primary resistance was determined at 12 weeks of treatment using criteria for progression that included serum prostate-specific antigen measurement, bone and computerized tomography imaging and symptom assessments. Acquired resistance was determined using the end point of time to treatment change (TTTC), defined as time from enrollment until change in treatment from progressive disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using logistic regression, Fisher‘s exact test, single and multivariate analyses. Cox regression models were utilized for determining association of genomic and transcriptomic alterations with TTTC. Results At 12 weeks, 32 patients in the cohort had progressed (nonresponders). Median study follow-up was 32.1 months by which time 58 patients had switched treatments due to progression. Median TTTC was 10.1 months (interquartile range: 4.4–24.1). Genes in the Wnt/s-catenin pathway were more frequently mutated and negative regulators of Wnt/s-catenin signaling were more frequently deleted or displayed reduced mRNA expression in nonresponders. Additionally, mRNA expression of cell cycle regulatory genes was increased in nonresponders. In multivariate models, increased cell cycle proliferation scores (≥50) were associated with shorter TTTC (hazard ratio = 2.11, 95% confidence interval: 1.17–3.80; P = 0.01). Conclusions Wnt/s-catenin pathway activation and increased cell cycle progression scores can serve as molecular markers for predicting resistance to AA/P therapy.

Fuchs endothelial corneal dystrophy (FECD) is a common cause for heritable visual loss in the elderly. Since the first description of an association between FECD and common polymorphisms situated within the transcription factor 4 (TCF4) gene, genetic and molecular studies have implicated an intronic CTG trinucleotide repeat (CTG18.1) expansion as a causal variant in the majority of FECD patients. To date, several non-mutually exclusive mechanisms have been proposed that drive and/or exacerbate the onset of disease. These mechanisms include (i) TCF4 dysregulation; (ii) toxic gain-of-function from TCF4 repeat-containing RNA; (iii) toxic gain-of-function from repeat-associated non-AUG dependent (RAN) translation; and (iv) somatic instability of CTG18.1. However, the relative contribution of these proposed mechanisms in disease pathogenesis is currently unknown. In this review, we summarise research implicating the repeat expansion in disease pathogenesis, define the phenotype-genotype correlations between FECD and CTG18.1 expansion, and provide an update on research tools that are available to study FECD as a trinucleotide repeat expansion disease. Furthermore, ongoing international research efforts to develop novel CTG18.1 expansion-mediated FECD therapeutics are highlighted and we provide a forward-thinking perspective on key unanswered questions that remain in the field., Highlights • FECD is a common, age-related corneal dystrophy. • The majority of cases are associated with expansion of a CTG repeat (CTG18.1). • FECD is the most common trinucleotide repeat expansion disease in humans. • Evidence supports multiple molecular mechanisms underlying the pathophysiology. • Novel CTG18.1-targeted therapeutics are in development.

Objective To describe the experience and outcome of performing whole-exome sequencing (WES) for resolution of patients on a diagnostic odyssey in the first 18 months of an individualized medicine clinic (IMC). Patients and Methods The IMC offered WES to physicians of Mayo Clinic practice for patients with suspected genetic disease. DNA specimens of the proband and relatives were submitted to WES laboratories. We developed the Genomic Odyssey Board with multidisciplinary expertise to determine the appropriateness for IMC services, review WES reports, and make the final decision about whether the exome findings explain the disease. This study took place from September 30, 2012, to March 30, 2014. Results In the first 18 consecutive months, the IMC received 82 consultation requests for patients on a diagnostic odyssey. The Genomic Odyssey Board deferred 7 cases and approved 75 cases to proceed with WES. Seventy-one patients met with an IMC genomic counselor. Fifty-one patients submitted specimens for WES testing, and the results have been received for all. There were 15 cases in which a diagnosis was made on the basis of WES findings; thus, the positive diagnostic yield of this practice was 29%. The mean cost per patient for this service was approximately $8000. Medicaid supported 27% of the patients, and 38% of patients received complete or partial insurance coverage. Conclusion The significant diagnostic yield, moderate cost, and notable health marketplace acceptance for WES compared with conventional genetic testing make the former method a rational diagnostic approach for patients on a diagnostic odyssey.

Background A 4-year-old boy born at 37 weeks' gestation with intrauterine growth retardation presented with developmental delay with pronounced language and gross motor delay, axial hypotonia, and dynamic hypertonia of the extremities. Investigations including the Minnesota Newborn Screen, thyroid stimulating hormone/thyroxin, and inborn errors of metabolism screening were negative. Cerebral magnetic resonance imaging and spectroscopy were normal. Genetic testing was negative for coagulopathy, Smith-Lemli-Opitz, fragile X, and Prader-Willi/Angelman syndromes. Whole genome array analysis was unremarkable. Methods Whole exome sequencing was performed through a commercial testing laboratory to elucidate the underlying etiology for the child's presentation. A de novo mutation was hypothesized. In attempt to establish pathogenicity of our candidate variant, cellular electrophysiologic functional analysis of the putative de novo mutation was performed using patch-clamp technology. Results Whole exome sequencing revealed a p.P1353L variant in the CACNA1A gene, which encodes for the α1-subunit of the brain-specific P/Q-type calcium channel (Ca V 2.1). This presynaptic high-voltage-gated channel couples neuronal excitation to the vesicular release of neurotransmitter and is implicated in several neurologic disorders. DNA Sanger sequencing confirmed that the de novo mutation was absent in both parents and present in the child only. Electrophysiologic analysis of P1353L-CACNA1A demonstrated near complete loss of function, with a 95% reduction in peak current density. Conclusions Whole exome sequencing coupled with cellular electrophysiologic functional analysis of a de novo CACNA1A missense mutation has elucidated the probable underlying pathophysiologic mechanism responsible for the child's phenotype. Genetic testing of CACNA1A in patients with congenital hypotonia and developmental delay may be warranted.

Objective To understand motivations, educational needs, and concerns of individuals contemplating whole-exome sequencing (WES) and determine what amount of genetic information might be obtained by sequencing a generally healthy cohort so as to more effectively counsel future patients. Patients and Methods From 2012 to 2014, 40 medically educated, generally healthy scientists at Mayo Clinic were invited to have WES conducted on a research basis; 26 agreed to be in a drawing from which 10 participants were selected. The study involved pre- and posttest genetic counseling and completion of 4 surveys related to the experience and outcomes. Whole-exome sequencing was conducted on DNA from blood from each person. Results Most variants (76,305 per person; range, 74,505-77,387) were known benign allelic variants, variants in genes of unknown function, or variants of uncertain significance in genes of known function. The results of suspected pathogenic/pathogenic variants in Mendelian disorders and pharmacogenomic variants were disclosed. The mean number of suspected pathogenic/pathogenic variants was 2.2 per person (range, 1-4). Four pharmacogenomic genes were included for reporting; variants were found in 9 of 10 participants. Conclusion This study provides data that may be useful in establishing reality-based patient expectations, outlines specific points to cover during counseling, and increases confidence in the feasibility of providing adequate preparation and counseling for WES in generally healthy individuals.

Purpose Hereditary hyperferritinemia cataract syndrome (HHCS), an autosomal-dominant disorder characterized by hyperferritinemia and bilateral cataracts, is caused by mutations in the iron-responsive element of the ferritin light chain ( FTL ) gene. The purpose of this study is to describe the genotypic and phenotypic manifestations of HHCS observed in 2 large sets of unrelated American families. Methods Forty-five patients were recruited from 2 unrelated families. Each underwent ophthalmological and general physical evaluation as well as laboratory testing of serum ferritin, iron, transferrin saturation, and total iron binding capacity. Serum DNA was evaluated for mutations by DNA amplification and sequencing of the FTL gene. Results Numerous cortical and nuclear white opacities in a stellate pattern occurred in 22 affected individuals and were the only clinical manifestation of HHCS. Of the 22, 16 (73%) demonstrated >1.00 D of astigmatism. Genetic analysis revealed mutation G32A in Pedigree 1 and mutation G32T in Pedigree 2, both heterozygous and located in the iron-responsive element of the ferritin light chain mRNA. Serum ferritin levels of affected subjects ranged from 555 to 2,453 μg/L (normal range, 24–336 μg/L male, 11–307 μg/L female), with greater ferritin levels and more severe cataracts associated with mutation G32A. Conclusions Most clinical and genetic findings from these families are consistent with previous reports of HHCS. Astigmatism, previously not associated with HHCS, was present in the majority. Ferritin levels and age of cataract surgery varied among subjects with both FTL gene mutations, suggesting that phenotypic variability is modulated by other genetic or environmental factors.

Membrane metallo-endopeptidase (MME), also known as neutral endopeptidase 24.11 (EC 3.4.24.11), is involved in the metabolism of natriuretic peptides that play a key role in modulating cardiac structure and function. Common genetic variation in MME has not been addressed by resequencing the gene using DNA from different ethnic populations. We set out to identify and functionally characterize common genetic variation in MME in three ethnic groups. DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence MME. Ninety polymorphisms, 65 novel, were identified, including 8 nonsynonymous single nucleotide polymorphisms (nsSNPs). Expression constructs for the nsSNPs were created and COS-1 cells were transfected with constructs for wild type (WT) and variant allozymes. Recombinant proteins were analyzed by quantitative Western blot analysis and by a one-step fluorometric assay. A significant reduction in enzyme activity (21% of WT) and immunoreactive protein (29% of WT) for the Val73 variant allozyme was observed. Proteasome-mediated degradation and autophagy participated in the degradation of this variant allozyme. The chaperone proteins, BiP and GRP94, were upregulated after transfection with Val73 MME, suggesting protein misfolding, compatible with conclusions based on the MME X-ray crystal structure. Multiple novel polymorphisms of MME were identified in three ethnic groups. The Val73 variant allozyme displayed a significant decrease in MME protein quantity and activity, with degradation mediated by both proteasome and autophagy pathways. This polymorphism could have a significant effect on the metabolism of natriuretic peptides.

Arsenic contaminates ground water worldwide. Methylation is an important reaction in the biotransformation of arsenic. We set out to study the pharmacogenetics of human arsenic methyltransferase (AS3MT, previously CYT19). After cloning the human AS3MT cDNA, we annotated the human gene and resequenced its 5′-flanking region, exons, and splice junctions using 60 DNA samples from African-American (AA) and 60 samples from Caucasian-American (CA) subjects. We observed 26 single nucleotide polymorphisms (SNPs), including 3 non-synonymous cSNPs, as well as a variable number of tandem repeats in exon 1 within an area encoding the cDNA 5′-untranslated region. The nonsynonymous cSNPs included T860C (M287T) with frequencies of 10.8 and 10% in AA and CA subjects, respectively, as well as C517T (A173W) in one AA and C917T (T306I) in one CA sample. Haplotype analysis showed that Ile306 was linked to Thr287, so this double variant allozyme was also studied functionally. After expression in COS-1 cells and correction for transfection efficiency, the Trp173 allozyme displayed 31%, Thr287 350%, Ile306 4.8%, and Thr287/Ile306 6.2% of the activity of the wild type (WT) allozyme, with 20, 190, 4.4, and 7.9% of the level of WT immunoreactive protein, respectively. Apparent Km values for S-adenosyl-l-methionine were 4.6, 3.1, and 11 μm for WT, Trp173, and Thr287 allozymes, with Km values for sodium arsenite with the same allozymes of 11.8, 8.9, and 4.5μm. The Ile306 and Thr287/Ile306 allozymes expressed too little activity for inclusion in the substrate kinetic studies. Expression of reporter gene constructs for the 5′-flanking region and the variable number of tandem repeats in the 5′-untranslated region demonstrated cell line-dependent variation in reporter gene expression, with shorter repeats associated with increased transcription in HepG2 cells. These results raise the possibility that inherited variation in AS3MT may contribute to variation in arsenic metabolism and, perhaps, arsenic-dependent carcinogenesis in humans.

Objective Imbalance between α 1 -antitrypsin and neutrophil elastase is an underlying cause of lung tissue damage that may create a favorable host environment for carcinogenesis. We conducted a case-control study to investigate whether genetic variations indicative of α 1 -antitrypsin deficiency (A1ATD) or an excess of neutrophil elastase modify lung cancer risk Design The case patients were 305 consecutively identified primary lung cancer patients, and the control subjects were 338 community residents. Protease inhibitor-1 (PI1), encoding α 1 -antitrypsin, was typed by an isoelectric focusing assay. Neutrophil elastase-2 (ELA2), encoding neutrophil elastase, was typed by two single-nucleotide polymorphism sites. Multivariable logistic regression models tested the independent and interactive effects of PI1, ELA2, tobacco smoke exposure, COPD, and family history of lung cancer Results Sex and ethnicity were comparable between case patients and control subjects, but case patients were more likely to be smokers, and to have a history of COPD, environmental tobacco smoke exposure, and a positive family history of lung cancer. Haplotype analysis indicated an overall strong association between the two ELA2 markers and lung cancer risk. Our best-fitting model showed significant and independent effects of the PI1-deficient allele (odds ratio [OR], 2.0; 95% confidence interval [CI], 1.4 to 3.0) and the ELA2 T-G haplotype (OR, 4.1; 95% CI, 1.9 to 8.9) on lung cancer risk, and an increased risk (OR, 2.6; 95% CI, 2.4 to 2.8) for individuals carrying both a PI1-deficient allele and a G-G haplotype Conclusions Genotypes indicative of A1ATD and/or an excess of neutrophil elastase are significantly associated with lung cancer risk. Our findings may provide opportunities to better understand the mechanisms of lung cancer development and risk reduction.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the high-energy "sulfate donor" for reactions catalyzed by sulfotransferase (SULT) enzymes. The strict requirement of SULTs for PAPS suggests that PAPS synthesis might influence the rate of sulfate conjugation. In humans, PAPS is synthesized from ATP and SO(4)(2-) by two isoforms of PAPS synthetase (PAPSS): PAPSS1 and PAPSS2. As a step toward pharmacogenetic studies, we have resequenced the entire coding sequence of the human PAPSS1 gene, including exon-intron splice junctions, using DNA samples from 60 Caucasian-American and 58 African-American subjects. Twenty-one genetic polymorphisms were observed-1 insertion-deletion event and 20 single nucleotide polymorphisms (SNPs)-including two non-synonymous coding SNPs (cSNPs) that altered the following amino acids: Arg333Cys and Glu531Gln. Twelve pairs of these polymorphisms were tightly linked, and a total of twelve unequivocal haplotypes could be identified-two that were common to both ethnic groups and ten that were ethnic-specific. The Arg333Cys polymorphism, with an allele frequency of 2.5%, was observed only in DNA samples from Caucasian subjects. The Glu531Gln polymorphism was rare, with only a single copy of that allele in a DNA sample from an African-American subject. Transient expression in mammalian cells showed that neither of the non-synonymous cSNPs resulted in a change in the basal level of enzyme activity measured under optimal assay conditions. However, the Glu531Gln polymorphism altered the substrate kinetic properties of the enzyme. The Gln531 variant allozyme had a 5-fold higher K(m) value for SO(4)(2-) than did the wild-type allozyme and displayed monophasic kinetics for Na(2)SO(4). The wild-type allozyme (Glu531) showed biphasic kinetics for that substrate. These observations represent a step toward testing the hypothesis that genetic variation in PAPS synthesis catalyzed by PAPSS1 might alter in vivo sulfate conjugation.

The draft sequence of the human genome was reported 2 years ago, and the task of filling gaps and polishing the sequence is nearing completion. However, despite this remarkable achievement, there is still no definitive assessment of the number of genes contained in the human genome. In part, this uncertainty reflects our growing understanding of the complexity and diversity of gene structure. Examples of complex gene structure are considered in the context of a discussion about the evolution of our understanding of gene structure and function.

Proteomics, simply defined, is the study of proteomes. More completely, proteomics is defined as the study of all proteins, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, in a given cell or organism within a given environment and at a specific stage in the cell cycle. Proteins carry out the biological functions encoded by genes; hence, once the initial stage of genome sequencing and gene discovery is completed, a study of the proteome must be undertaken to address fundamental biological questions. The 3 broad areas are expression proteomics, which catalogues the relative abundance of proteins; cell-mapping or cellular proteomics, which delineates functional protein-protein interactions and organelle-specific protein distribution; and structural proteomics, which characterizes the 3-dimensional structure of proteins. With these approaches, proteins are studied on a global scale using a synergistic combination of powerful, high-throughput technologies, including 2-dimensional polyacrylamide gel electrophoresis, mass spectrometry, multidimensional liquid chromatography, and bioinformatics. Mass spectrometry, which provides highly accurate molecular mass measurements, has emerged as the analytical technology of choice for protein identification, characterization, and sequencing. This task has been made considerably easier with the availability of complete, nonredundant, and annotated genome sequence databases for many organisms. This article reviews the area of expression proteomics.

Genomics has been defined as the comprehensive study of whole sets of genes, gene products, and their interactions as opposed to the study of single genes or proteins. Microarray technology is one of many novel tools that are allowing global and high-throughput analysis of genes and gene products. In addition to an introduction on underlying principles, the current review focuses on the use of both complementary DNA and oligodeoxynucleotide microarrays in gene expression analysis. Genome-wide experiments generate a massive amount of data points that require systematic methods of analysis to extract biologically useful information. Accordingly, the current educational communication discusses different methods of data analysis, including supervised and unsupervised clustering algorithms. Illustrative clinical examples show clinical applications, including (1) identification of candidate genes or pathological pathways (ie, elucidation of pathogenesis); (2) identification of "new" molecular classes of diseases that may be relevant in disease reclassification, prognostication, and treatment selection (ie, class discovery); and (3) use of expression profiles of known disease classes to predict diagnosis and classification of unknown samples (ie, class prediction). The current review should serve as an introduction to the subject for clinician investigators, physicians and medical scientists in training, practicing clinicians, and other students of medicine.

In comparison with most other disciplines of science, the field of genetics is still in its youth. The majority of scientific work in genetics has been done in the past 150 years. The successful preliminary sequencing of the human genome was announced in 2001. Nonetheless, interest in heredity and in other concepts within the field of genetics has existed since the beginning of humanity. This article provides an account of the history of genetics, spanning from humankind's initial attempts to understand and influence heredity, to the early scientific work in the field of genetics, and subsequently to the advancements in modern genetics. Additionally, the Human Genome Project is summarized, from inception to publication of the 'first draft" of the human genome sequence.

A portion of the 5'-flanking region of murine acetylcholinesterase was cloned from genomic DNA by 5'-rapid amplification of genomic ends, identified in a mouse genomic library, and sequenced. Multiple potential binding sites for universal and tissue-specific transcription factors were suggestive of a promoter region within this DNA sequence. Potential promoter activity was confirmed by coupling the new sequence to the open reading frame of a luciferase reporter gene in transient expression experiments with nerve and muscle cells. 5'-Rapid amplification of cDNA ends with templates from multiple sources revealed a novel transcription start site (at position -626, relative to translation start), located 32 bases downstream from a TATAA sequence. This start site appeared to mark a novel exon (1a) comprising 291 base pairs between positions -335 and -626, relative to the translation start. Supporting this conclusion, polymerase chain reactions with cDNA from mouse brain, heart, and other tissues, consistently amplified a transcript containing the exon 1a sequence fused to the invariant sequence beginning at position -22 in exon 2, but lacking exon 1. Northern blot analyses confirmed the in vivo expression of exon 1a-containing transcripts, especially in heart, brain, liver, and kidney. These results indicate that the murine acetylcholinesterase gene has a functioning alternative promoter that may influence expression of acetylcholinesterase in certain tissues.

TGFbeta-inducible early gene (TIEG) and early growth response alpha (EGRalpha) are putative transcription factors based on homology to known zinc finger proteins SP1, EGR1, BTEB, and Wilm tumor. Here we report that TIEG and EGRalpha are expressed from alternative promoters of the same gene. The TIEG/EGRalpha gene spans 8 kb and contains five exons. Use of alternative first exons results in TIEG having 12 unique amino acids on its N-terminus. Computer analysis of the 5' upstream regions of either TIEG (exon 1a) or EGRalpha (exon 1b) does not identify a TATA box or initiator sequencebut shows consensus sequence similarities to binding sites for several transcription factors including SP1,JunB, and aromatic hydrocarbon/receptor-ligand complexes. Analysis of constructs containing 5'-flanking regions show that both the TIEG and the EGRalpha promoters have significant activity in human fetal osteoblast cells. Northern analysis of mRNA from various human tissues and several cell lines reveals that TIEG is the predominant transcript produced and regulated by growth factors from the TIEG/EGRalpha gene.

The GP1G gene of the guinea pig codes for three of the four abundant seminal vesicle secretory proteins produced in this species. This gene is expressed at highest efficiency in the seminal vesicle (SV) from a promoter that contains a canonical TATA box and CCAAT box. However, GP1G gene transcripts and proteins have also been identified in other tissues. To investigate the structure of GP1G transcripts produced in the testis, cDNA clones were isolated by screening a testis library. Three unique cDNAs (TSM1-3) were isolated. Each of these clones contained a 3'-untranslated region (UTR) and coding region identical to that of the seminal vesicle transcript. However, the 5'-UTRs of the testis transcripts were significantly longer than that found on the SV mRNA (416-646 nucleotides compared with only 23 nucleotides for the SV). Each of these alternatively spliced 5'-UTRs incorporated the SV promoter elements into transcribed sequence, and each contained multiple upstream AUG codons predicted to abolish translation of the major open reading frame. Nevertheless, each of the testis transcripts was capable of directing the synthesis of GP1G-related proteins in vitro. Analysis of the translation products suggests that the extended 5'-UTR of the testis transcripts regulate both the choice of translation start site and the efficiency of translation in this system. Western blot analysis of testis proteins revealed that the protein products of GP1G are also synthesized by the testis in vivo.

The GP1G gene codes for three of the four abundant androgen-regulated secretory proteins produced by the guinea pig seminal vesicle. Sequencing of the entire 6.3-kilobase gene and comparison with other mammalian seminal vesicle secretory protein genes reveals a common three-exon, two-intron organization. However, significant sequence similarity between this group of genes is largely limited to their 5′-flanking regions and first exons, which code almost exclusively for signal peptides in each case. The first intron of GP1G does contain a region with high similarity to the coding exon of a human seminal vesicle secretory protein gene, semenogelin II. The 3′ half of the GP1G gene appears to share a common ancestry with the human SKALP/elafin gene. Sequences related to the elafin promoter, coding, untranslated regions, and introns are clearly identifiable within the GP1G sequence. The elafin gene codes for a serine protease inhibitor and is expressed in a variety of different human tissues. To determine if the GP1G gene was also active outside of the seminal vesicle, RNA from a variety of guinea pig tissues was hybridized to a GP1G cDNA probe. At least three novel RNA bands hybridizing to the GP1G probe were detected in testis RNA samples, and GP1G-related mRNAs were also found in other tissues. These data suggest that these seminal vesicle secretory proteins may have functional roles outside the reproductive system.

Background & Aims: Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product. Methods: Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient. Results: Ninety-three percent of the patient's CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% ± 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient. Conclusions: Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans -acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients.

We expressed milligram amounts of functional human 1,25-dihydroxyvitamin D3 receptor in a bacterial expression system in which the cloned cDNA for the hVDR was expressed under the control of bacterial T7 polymerase. The hVDR protein comprised approximately 60% of total bacterial protein. It migrated on polyacrylamide-sodium dodecyl sulfate gels with an Mr of 48,000. It had the predicted amino acid composition and amino acid sequence analysis. The expressed protein was bound by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with a Kd in the nanomolar range. It sedimented on sucrose density gradients at 3.5S. Furthermore, the expressed protein bound to the osteocalcin vitamin D response element (VDRE) as assessed by a gel mobility shift assay. The expression of large amounts of hVDR protein should allow for the use of this protein in structure-function and x-ray crystallography studies.

Using the polymerase chain reaction (PCR), we identified RNA transcripts for two distinct classes of neuronal-type voltage-gated Ca2+ channels (VGCC) in a prototypic small cell lung carcinoma (SCLC) cell line, SCC-9. Oligonucleotide primers were designed to encode amino acid sequences common to alpha 1-subunits of known neuronal VGCC classes. Sequencing of complementary DNA (cDNA) clones derived from two independent PCR products revealed that one corresponded to a brain class A VGCC fragment predicted to encode a P-type VGCC (insensitive to dihydropyridines and omega-conotoxin) characteristic of cerebellar Purkinje cells but not previously identified in humans. The second PCR product was identical (except for one conservative nucleotide difference) to a fragment of the class D VGCC of neurons and neuroendocrine cells, which encodes an L-type VGCC (sensitive to dihydropyridines). By Northern blot analyses, both cDNAs hybridized to messenger RNAs (mRNAs) obtained from SCC-9; class D hybridized additionally to human cerebral cortical mRNA, but neither hybridized to mRNA from the skeletal muscle cell line TE671. Although no cDNA corresponding to class B VGCC (N-type) was identified, SCLCs are known to express VGCC that are sensitive to omega-conotoxin and coprecipitate with 125I-labeled-omega-conotoxin when complexed with serum IgG from patients with the Lambert-Eaton myasthenic syndrome. The multiple classes of neuronal-type VGCC expressed in SCLC could conceivably have both unique and related antigenic determinants that may give rise to antineuronal autoimmune responses. This would account for a spectrum of paraneoplastic neurologic disorders including the Lambert-Eaton syndrome and subacute cerebellar degeneration.

The 5'-upstream region of the small nuclear ribonucleoprotein E protein (snRNP E) gene has multiple sequence similarities to elements found to be integral in the transcriptional regulation of some mouse ribosomal protein genes (G + C block, CTTCCG motifs) as well as U1 snRNA genes (U1 "B," U1 "D," and SPH enhancers). Furthermore, the immediate upstream region of the snRNP E protein gene lacks typical TATA and CAAT sequence motifs but contains one copy of a GC box characteristic of a housekeeping promoter. By transfection of constructs containing various amounts of the 5'-upstream region of the snRNP E protein gene linked to the bacterial gene for chloramphenicol acetyl transferase (CAT), we determined that the 5' boundary of sequence needed for transcription was within the first 153 nucleotides upstream of the transcription start site. Further deletions to within 51 base pairs reduced CAT expression by 2.5-fold. Mutational analysis within this 51-base pair region revealed that direct repeats of the hexamer CTTCCG were essential for CAT expression. Gel shift assays and DNase I footprinting experiments confirmed this conclusion by demonstrating specific binding of proteins to the CTTCCG motifs. This suggests that the direct repeats of the CTTCCG hexamer sequence play a key role in transcriptional regulation of the snRNP E protein gene.

Small nuclear ribonucleoproteins (snRNPs), which are composed of various U RNAs and several proteins, are components of the mRNA splicing apparatus. The snRNP protein E is encoded by a multigene family which consists of a single expressed gene and several processed pseudogenes. We have used somatic cell hybridization, in situ hybridization, and linkage analysis to both physically and genetically map the expressed E protein gene to human chromosome 1q25–43, with the most probable location being band 1q32. In addition to the snRNP E protein gene, two other snRNP components—the U1 RNA true multigene family and a group of class I U1 pseudogenes—are located on human chromosome 1.

We recently identified two CMS patients with CNS involvement. Patient 1 is a16-year-old mentally retarded girl with severe generalized CMS since infancy. One of her siblings is also affected and has autistic features. Patient 2 is a 14-year-old girl with mild cognitive deficits and progressive limb-girdle CMS since infancy. Both respond poorly to anti-AChE therapy. Intercostal muscle specimens in both show small tubular aggregates in type 2 fibers, type1 fiber atrophy, and a vacuolar myopathy with autophagic features. Endplate studies reveal that quantal release, postsynaptic response to acetylcholine, and endplate AChR content are reduced to ∼50% of normal. Quantitative EM of 65 endplate regions shows hypoplastic endplates, very small nerve terminals, and poorly differentiated postsynaptic regions. Neither patient harbors mutations in currently recognized CMS disease genes but exome sequencing in each identified two heteroallelic mutations in DPAGT1 coding for dolichyl-phosphate N-acetylglucosamine phosphotransferase, an enzyme subserving protein N-glycosylation. Immunoblots of muscle extracts probed by two different antibodies demonstrates reduced to absent glycosylation of ∼70 kDa proteins in Patient 1 but not in Patient 2. We conclude that DPAGT1 mutations result in both pre- and postsynaptic structural and electrophysiologic abnormalities. We hypothesize that hypoglycosylation of synapse-specific proteins causes defects in motor as well as central synapses.

Type and Scope of Book: A comprehensive multiauthored text that focuses on the impact of the evolving field of pharmacogenomics on medicine and society.

Guinea pig seminal vesicle epithelium is an androgen-dependent tissue composed of tall columnar cells which synthesize four major secretory proteins designated SVP-1-4. Castration promptly causes the seminal vesicle epithelium cells to undergo major morphological changes. By the fifth day after castration, cell number decreases to 12% of normal, and the surviving cells are greatly involuted structurally. An injection of testosterone was sufficient to stimulate individual cell growth to the normal tall columnar configuration by 48 h without increasing cell number. Cell growth following testosterone was preceded by a 4-fold increase between 0-12 h in total cellular RNA signifying a large increase in rRNAs. To aid in the analysis of gene expression during androgen repletion, we identified a cDNA plasmid clone representing the 3'-end of the mRNA of SVP-4. The clone pAT 76 was identified by hybrid-select translation and characterization of the product of in vitro translation. The steady-state levels of SVP-4 mRNA in blots of total cellular RNA increased between 0-24 h; this appeared to be due mainly to a general nonselective increase in all or most of the abundant mRNAs. We demonstrated by in vitro translation studies that the corresponding transcript was still present during the 5th and 36th days of castration without having undergone a selective loss in abundance and during hormone repletion without undergoing a selective increase. Testosterone exerted an early and more conspicuous effect on rRNA synthesis. Whether direct or indirect this may be a key event underlying the ultimate action of testosterone, namely maintenance of cell structure.

Steroid hormones have been shown to have highly differential effects on the expression of abundant cell-specific protein genes in a multitude of model tissues. In rat seminal vesicle, for example, DNA clones representing two major secretory protein genes have been used to show that both of the genes are differentially regulated by androgen. In this paper, we have examined the effects of androgen on the transcription of two major secretory protein genes in guinea pig seminal vesicle epithelium. Nuclear run-off experiments were used to show that castration of the adult resulted in a 3-fold decrease in total transcription activity. Surprisingly, the decrease in total transcriptional activity was not reflected in a differential decrease in the transcriptional activity of the two major secretory protein genes. When the effects of castration on the transcriptional activity of the major secretory protein genes were compared to the effects on other genes, it was found that the transcriptional activity of each gene examined was decreased by the same magnitude as the major secretory protein genes. Similarly, the transcriptional activity of every gene examined increased by the same magnitude as the major secretory protein genes during hormone repletion of the castrated adult. Thus, in contrast to the differential effects of steroids on the transcription of abundant cell-specific proteins in many other steroid-dependent tissues, the transcription of major secretory proteins in guinea pig seminal vesicle epithelium appears to be regulated in parallel with many other genes. The generalized effects of androgen on transcriptional activity could account for the generalized effects of androgen on seminal vesicle epithelial cell structure and function.

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.

The sequence of a complementary DNA clone coding for a human autoimmune antigen has been determined. This DNA sequence predicts the amino acid sequence of a small protein ("E") which is associated with small nuclear RNA in human cells. Analysis of the predicted protein sequence suggests that the E protein is not closely related to other nucleic acid binding proteins. Screening of a human genomic DNA library has led to the isolation of several members of the E protein multigene family. Sequence analysis of one member of this family reveals that it is flanked by direct repeats and contains several mutations. One of these mutations, an insertion, terminates the long open reading frame. These features are compatible with the designation of this sequence as a processed pseudogene.

cDNAs coding for a plasma membrane Ca2+ pump were isolated from a human teratoma library and sequenced. The translated sequence contained 1,220 amino acids with a calculated molecular weight of 134,683. All regions of functional importance known from other ion-transporting ATPases could be identified. The translated sequence also contained, near the carboxyl terminus, the calmodulin-binding domain and two domains which are very rich in glutamic acid and aspartic acid. These two domains resemble calmodulin somewhat and one of them may play a role in the binding of Ca2+. The enzyme also contains domains rich in serine and threonine, one of which has a sequence matching those of good cAMP-dependent protein kinase substrates. The carboxyl-terminal region is important for regulation by calmodulin, proteolysis, and phosphorylation. Near the amino terminus are two domains which are very rich in lysine and glutamic acid, as well as two domains resembling EF hands, one of which also has some resemblance to calmodulin. Comparison of the cloned sequence with peptide sequences from the erythrocyte Ca2+ pump showed that the two proteins have a very high proportion of identical residues but are not 100% identical, indicating that they represent different isozymes.