Assessing the mitochondrial DNA diversity of the Chagas disease vector Triatoma sordida (Hemiptera: Reduviidae)

Triatoma sordida is a species that transmits Trypanosoma cruzi to humans. In Brazil, T. sordida currently deserves special attention because of its wide distribution, tendency to invade domestic environments and vectorial competence. For the planning and execution of control protocols to be effective against Triatominae, they must consider its population structure. In this context, this study aimed to characterise the genetic variability of T. sordida populations collected in areas with persistent infestations from Minas Gerais, Brazil. Levels of genetic variation and population structure were determined in peridomestic T. sordida by sequencing a polymorphic region of the mitochondrial cytochrome b gene. Low nucleotide and haplotype diversity were observed for all 14 sampled areas; π values ranged from 0.002-0.006. Most obtained haplotypes occurred at low frequencies, and some were exclusive to only one of the studied populations. Interpopulation genetic diversity analysis revealed strong genetic structuring. Furthermore, the genetic variability of Brazilian populations is small compared to that of Argentinean and Bolivian specimens. The possible factors related to the reduced genetic variability and strong genetic structuring obtained for studied populations are discussed in this paper.

Disease in the dark: molecular characterization of Polychromophilus murinus in temperate zone bats revealed a worldwide distribution of this malaria-like disease.

For a better understanding of the complex coevolutionary processes between hosts and parasites, accurate identification of the actors involved in the interaction is of fundamental importance. Blood parasites of the Order Haemosporidia, responsible for malaria, have become the focus of a broad range of studies in evolutionary biology. Interestingly, molecular-based studies on avian malaria have revealed much higher species diversity than previously inferred with morphology. Meanwhile, studies on bat haemosporidian have been largely neglected. In Europe, only one genus (Polychromophilus) and two species have been morphologically described. To evaluate the presence of potential cryptic species and parasite prevalence, we undertook a molecular characterization of Polychromophilus in temperate zone bats. We used a nested-PCR approach on the cytochrome b mitochondrial gene to detect the presence of parasites in 237 bats belonging to four different species and in the dipteran bat fly Nycteribia kolenatii, previously described as being the vector of Polychromophilus. Polychromophilus murinus was found in the four bat species and in the insect vector with prevalence ranging from 4% for Myotis myotis to 51% for M. daubentoni. By sequencing 682 bp, we then investigated the phylogenetic relationships of Polychromophilus to other published malarial lineages. Seven haplotypes were found, all very closely related, suggesting the presence of a single species in our samples. These haplotypes formed a well-defined clade together with Haemosporidia of tropical bats, revealing a worldwide distribution of this parasite mostly neglected by malarial studies since the 1980s.

High-level molecular diversity of copper-zinc superoxide dismutase genes among and within species of arbuscular Mycorrhizal fungi.

In the ecologically important arbuscular mycorrhizal fungi (AMF), Sod1 encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at the Sod1 locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of the Sod1 gene in six AMF species, as well as 14 Glomus intraradices isolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. The Sod1 gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy in G. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates of G. intraradices from a natural population harbor very divergent orthologous Sod1 sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.

Early onset collagen VI myopathies: Genetic and clinical correlations.

OBJECTIVE: Mutations in the genes encoding the extracellular matrix protein collagen VI (ColVI) cause a spectrum of disorders with variable inheritance including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate phenotypes. We extensively characterized, at the clinical, cellular, and molecular levels, 49 patients with onset in the first 2 years of life to investigate genotype-phenotype correlations. METHODS: Patients were classified into 3 groups: early-severe (18%), moderate-progressive (53%), and mild (29%). ColVI secretion was analyzed in patient-derived skin fibroblasts. Chain-specific transcript levels were quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and mutation identification was performed by sequencing of complementary DNA. RESULTS: ColVI secretion was altered in all fibroblast cultures studied. We identified 56 mutations, mostly novel and private. Dominant de novo mutations were detected in 61% of the cases. Importantly, mutations causing premature termination codons (PTCs) or in-frame insertions strikingly destabilized the corresponding transcripts. Homozygous PTC-causing mutations in the triple helix domains led to the most severe phenotypes (ambulation never achieved), whereas dominant de novo in-frame exon skipping and glycine missense mutations were identified in patients of the moderate-progressive group (loss of ambulation). INTERPRETATION: This work emphasizes that the diagnosis of early onset ColVI myopathies is arduous and time-consuming, and demonstrates that quantitative RT-PCR is a helpful tool for the identification of some mutation-bearing genes. Moreover, the clinical classification proposed allowed genotype-phenotype relationships to be explored, and may be useful in the design of future clinical trials.

Emergence of secretion-defective sublines of Pseudomonas aeruginosa PAO1 resulting from spontaneous mutations in the vfr global regulatory gene.

Pseudomonas aeruginosa undergoes spontaneous mutation that impairs secretion of several extracellular enzymes during extended cultivation in vitro in rich media, as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum-sensing regulatory gene lasR. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum-sensing control. Using generalized transduction, we mapped the secretion defect to the vfr gene, which is known to control positively the expression of the lasR gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the vfr mutation. In a reconstruction experiment conducted with a 1:1 mixture of wild-type strain PAO1 and a vfr mutant of PAO1, we observed that the vfr mutant had a selective advantage over the wild type after growth in static culture for 4 days. Under these conditions, spontaneous vfr emerged in a strain PAO1 population after four growth cycles, and these mutants accounted for more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to P. aeruginosa under certain environmental conditions.

Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis.

Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter -15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter -15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.

Infective Endocarditis in a Finnish Teaching Hospital. 25 Years of Experience of Adult Patients

Infektiivinen endokardiitti yliopistollisessa keskussairaalassa vuosina 1980-2004 hoidetuilla aikuispotilailla Tausta: Infektiivinen endokardiitti on edelleen vakava sairaus. Huolimatta siitä, että taudin diagnostiikka ja hoito ovat kehittyneet, siihen liittyy edelleen merkittävää sairastuvuutta ja kuolleisuutta. Endokardiitin taudinkuvassa on viime vuosina tapahtunut muutoksia monissa maissa. Tavoitteet: Tutkia endokardiitin kliinista kuvaa ja ennustetta suomalaisessa yliopistosairaalassa vuosina 1980-2004 endokardiitin vuoksi hoidetuilla aikuispotilailla. Aineisto: Osatyössä I endokardiitin todennäköisyyttä analysoitiin 222:lla vuosina 1980-1995 endokardiittiepäilyn vuoksi hoidetulla potilaalla käyttäen apuna sekä Duken että von Reyn diagnostisia kriteereitä. Osatyössä II tutkittiin endokardiittiin liittyviä neurologisia komplikaatioita 218 varmassa tai mahdollisessa endokardiittiepisodissa. Osatyössä III tutkittiin seerumin C-reaktiivisen proteiinin (CRP) käyttökelpoisuutta hoitovasteen arvioinnissa 134:ssä varmaksi luokitellussa endokardiittiepisodissa. Osatyössä IV tutkittiin yleisbakteeri-PCRmenetelmän käyttökelpoisuutta etiologisessa diagnostiikassa 56:lla endokardiittiepäilyn vuoksi leikatulla potilaalla. Osatöissä V ja VI analysoitiin kaikki vuosina 1980-2004 hoidetut 303 endokardiittipotilasta lyhytaikais- ja 1-vuotisennusteen suhteen sekä tutkittiin endokardiitin taudinkuvassa tapahtuneita muutoksia sairaalassamme. Tulokset: Duken kriteerit osoittautuivat von Reyn kriteereitä herkemmiksi endokardiitin diagnostiikassa: 243 tutkitusta episodista 114 luokiteltiin varmoiksi endokardiiteiksi Duken kriteereillä, kun vastaavasti ainoastaan 64 luoteltiin varmoiksi von Reyn kriteereillä (p<0.001). Lisäksi peräti 115 episodissa endokardiitin diagnoosi hylättiin von Reyn kriteereillä, kun diagnoosi hylättiin Duken kriteereillä ainoastaan 37 episodissa (p<0.001). Neurologinen komplikaatio ilmeni ennen mikrobilääkehoidon aloittamista 76 %:ssa episodeja ollen ensimmäinen oire 47 %:ssa. Kuolema oli merkitsevästi yhteydessä neurologisiin komplikaatioihin. Hoitovastetta seurattaessa seerumin CRP:n lasku oli merkitsevästi nopeampaa komplikaatioitta toipuvilla potilailla kuin niillä, joille kehittyi komplikaatioita tai jotka menehtyivät tautiinsa. PCR-tutkimus poistetusta läpästä antoi ainoana menetelmänä etiologisen diagnoosin neljässä tapauksessa (2 stafylokokkilajia, 1 Streptococcus bovis,1 Bartonella quintana), joissa kaikissa mikrobilääkehoito oli ollut käytössä ennen näytteiden ottamista. Koko aineistossa kahden läpän infektio tai neurologisten komplikaatioiden, perifeeristen embolioiden tai sydämen vajaatoiminnan kehittyminen ennustivat sekä sairaalakuolleisuutta että 1-vuotiskuolleisuutta, kun taas ≥65 vuoden ikä ja sydämen ultraäänitutkimuksessa todettu vegetaatio tai Duken luokittelun mukainen pääkriteeri ennustivat kuolemaa vuoden sisällä. Korkea CRP-taso sairaalaan tullessa ennusti sekä sairaalakuolleisuutta että 1-vuotiskuolleisuutta. Huumeiden käyttäjien endokardiitit lisääntyivät tutkimusaikana merkitsevästi (p<0.001). Päätelmät: Tässä työssä vahvistetaan Duken kriteerien käyttökelpoisuus endokardiitin diagnostiikassa. Lisäksi vahvistui käsitys, että nopea diagnoosi ja mikrobilääkehoidon aloittaminen ovat parhaat keinot ehkäistä neurologisia komplikaatioita ja parantaa endokardiittipotilaiden ennustetta. CRP:n normalisoituminen on endokardiittipotilailla hyvän ennusteen merkki. Suoraan läppäkudoksesta tehty PCR-tutkimus on hyödyllinen, kun taudin aiheuttaja on kasvuominaisuuksiltaan vaativa tai potilas on saanut mikrobilääkehoitoa ennen viljelynäytteiden ottamista. Muutamat aiemmissa tutkimuksissa todetut huonon ennusteen merkit ennustavat huonoa ennustetta myös tämän tutkimuksen potilailla. Uutena löydöksenä ilmeni, että korkea CRP-arvo sairaalaan tullessa merkitsee sekä huonoa lyhyt- että pitkäaikaisennustetta. Huumeiden käyttäjien endokardiittien ilmaantuminen on tärkein epidemiologinen muutos 25 vuoden tutkimusaikana.

Recombination in Glomus intraradices, a supposed ancient asexual arbuscular mycorrhizal fungus

Background: Arbuscular mycorrhizal fungi (AMF) are important symbionts of most plant species, promoting plant diversity and productivity. This symbiosis is thought to have contributed to the early colonisation of land by plants. Morphological stasis over 400 million years and the lack of an observed sexual stage in any member of the phylum Glomeromycota led to the controversial suggestion of AMF being ancients asexuals. Evidence for recombination in AMF is contradictory. Results: We addressed the question of recombination in the AMF Glomus intraradices by sequencing 11 polymorphic nuclear loci in 40 morphologically identical isolates from one field. Phylogenetic relationships among genotypes showed a reticulate network pattern providing a rationale to test for recombination. Five statistical tests predicted multiple recombinant regions in the genome of a core set of isolates. In contrast, five clonal lineages had fixed a large number of differences. Conclusion: Our data show that AMF from one field have undergone recombination but that clonal lineages coexist. This finding has important consequences for understanding AMF evolution, co-evolution of AMF and plants and highlights the potential for commercially introduced AMF inoculum recombining with existing local populations. Finally, our results reconcile seemingly contradictory studies on whether AMF are clonal or form recombining populations.

Long-distance wolf recolonization of France and Switzerland inferred from non-invasive genetic sampling over a period of 10 years

In the early 1900s, the wolf (Canis lupus) was extirpated from France and Switzerland. There is growing evidence that the species is presently recolonizing these countries in the western Alps. By sequencing the mitochondrial DNA (mtDNA) control region of various samples mainly collected in the field (scats, hairs, regurgitates, blood or tissue; n = 292), we could (1) develop a non-invasive method enabling the unambiguous attribution of these samples to wolf, fox (Vulpes vulpes) or dog (Canis familiaris), among others; (2) demonstrate that Italian, French and Swiss wolves share the same mtDNA haplotype, a haplotype that has never been found in any other wolf population world-wide. Combined together, field and genetic data collected over 10 years corroborate the scenario of a natural expansion of wolves from the Italian source population. Furthermore, such a genetic approach is of conservation significance, since it has important consequences for management decisions. This first long-term report using non-invasive sampling demonstrates that long-distance dispersers are common, supporting the hypothesis that individuals may often attempt to colonize far from their native pack, even in the absence of suitable corridors across habitats characterized by intense human activities.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Revisit of multiple epiphyseal dysplasia: ethnic difference in genotypes and comparison of radiographic features linked to the COMP and MATN3 genes.

Multiple epiphyseal dysplasia (MED) is a genetically heterogeneous group of diseases characterized by variable degrees of epiphyseal abnormality primarily involving the hip and knee joints. The purpose of this study was to investigate the frequency of mutations in individuals with a clinical and radiographic diagnosis of MED and to test the hypothesis that characteristic radiological findings may be helpful in predicting the gene responsible. The radiographs of 74 Korean patients were evaluated by a panel of skeletal dysplasia experts. Six genes known to be associated with MED (COMP, MATN3, COL9A1, COL9A2, COL9A3, and DTDST) were screened by sequencing. Mutations were found in 55 of the 63 patients (87%). MATN3 mutations were found in 30 patients (55%), followed by COMP mutations in 23 (41%), and COL9A2 and DTDST mutations in one patient (2%) each. Comparisons of radiographic findings in patients with COMP and MATN3 mutations showed that albeit marked abnormalities in hip and knee joints were observed in both groups, the degree of involvement and the morphology of dysplastic epiphyses differed markedly. The contour of the pelvic acetabulum, the presence of metaphyseal vertical striations, and/or the brachydactyly of the hand were also found to be highly correlated with the genotypes. The study confirms that MATN3 and COMP are the genes most frequently responsible for MED and that subtle radiographic signs may give precious indications on which gene(s) should be prioritized for mutational screening in a given individual.

Identification and characterization of pathogenic Pestalotiopsis species to pecan tree in Brazil

The objective of this work was to characterize and cluster isolates of Pestalotiopsis species and to identify those that are pathogenic to pecan, based on morphological and molecular characters. Pestalotiopsis spp. isolates were identified by sequencing the internal transcribed spacer (ITS) and β?tubulin regions. Identification methods were compared to indicate the key morphological characters for species characterization. Thirteen isolates were used for the pathogenicity tests. Morphological characterization was performed using the following variables: mycelial growth rate, sporulation, colony pigmentation, and conidial length and width. Ten pathogenic isolates were identified, three as -tubulin regions. Identification methods were compared to indicate the key morphological characters for species characterization. Thirteen isolates were used for the pathogenicity tests. Morphological characterization was performed using the following variables: mycelial growth rate, sporulation, colony pigmentation, and conidial length and width. Ten pathogenic isolates were identified, three as Pestalotiopsis clavispora and three as P. cocculi. The other isolates remained as an undefined species. The morphological characters were efficient for an initial separation of the isolates, which were grouped according to differences at species level, mainly colony diameter, which was identified as an important morphological describer. Beta-tubulin gene sequencing was less informative than the ITS region sequencing for species identification.

Prevalence and diversity of Chlamydiales in Swiss ruminant farms.

Chlamydia and Chlamydia-related bacteria are known to infect various organisms and may cause a wide range of diseases, especially in ruminants. To gain insight into the prevalence of these bacteria in the ruminant environment, we applied a pan-Chlamydiales PCR followed by sequencing to 72 ruminant environmental samples from water, feed bunks and floors. Chlamydiales from four family-level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant farms. Parachlamydiaceae were detected in all three types of environmental samples and was the most abundant family-level taxon (60%). In contrast, only one bacterium from each of the following family-level lineages was identified: Chlamydiaceae, Criblamydiaceae and Simkaniaceae. The observed high prevalence of Parachlamydiaceae in water samples may suggest water as the main source of contamination for ruminants as well as their environment due to spoilage. The absence of reported infections in the investigated ruminant farms might indicate that either detected Chlamydiales are of reduced pathogenicity or infective doses have not been reached.

Newly described human polyomaviruses Merkel Cell, KI and WU are present in urban sewage and may represent potential environmental contaminants

Recently, three new polyomaviruses (KI, WU and Merkel cell polyomavirus) have been reported to infect humans. It has also been suggested that lymphotropic polyomavirus, a virus of simian origin, infects humans. KI and WU polyomaviruses have been detected mainly in specimens from the respiratory tract while Merkel cell polyomavirus has been described in a very high percentage of Merkel cell carcinomas. The distribution, excretion level and transmission routes of these viruses remain unknown. Here we analyzed the presence and characteristics of newly described human polyomaviruses in urban sewage and river water in order to assess the excretion level and the potential role of water as a route of transmission of these viruses. Nested-PCR assays were designed for the sensitive detection of the viruses studied and the amplicons obtained were confirmed by sequencing analysis. The viruses were concentrated following a methodology previously developed for the detection of JC and BK human polyomaviruses in environmental samples. JC polyomavirus and human adenoviruses were used as markers of human contamination in the samples. Merkel cell polyomavirus was detected in 7/8 urban sewage samples collected and in 2/7 river water samples. Also one urine sample from a pregnant woman, out of 4 samples analyzed, was positive for this virus. KI and WU polyomaviruses were identified in 1/8 and 2/8 sewage samples respectively. The viral strains detected were highly homologous with other strains reported from several other geographical areas. Lymphotropic polyomavirus was not detected in any of the 13 sewage neither in 9 biosolid/sludge samples analyzed. This is the first description of a virus isolated from sewage and river water with a strong association with cancer. Our data indicate that the Merkel cell polyomavirus is prevalent in the population and that it may be disseminated through the fecal/urine contamination of water. The procedure developed may constitute a useful tool for studying the excreted strains, prevalence and transmission of these recently described polyomaviruses.

Buried in the Middle but Guilty: Intronic Mutations in the TCIRG1 Gene Cause Human Autosomal Recessive Osteopetrosis.

Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease with genotypic and phenotypic heterogeneity, sometimes translating into delayed diagnosis and treatment. In particular, cases of intermediate severity often constitute a diagnostic challenge and represent good candidates for exome sequencing. Here, we describe the tortuous path to identification of the molecular defect in two siblings, in which osteopetrosis diagnosed in early childhood followed a milder course, allowing them to reach the adult age in relatively good conditions with no specific therapy. No clearly pathogenic mutation was identified either with standard amplification and resequencing protocols or with exome sequencing analysis. While evaluating the possible impact of a 3'UTR variant on the TCIRG1 expression, we found a novel single nucleotide change buried in the middle of intron 15 of the TCIRG1 gene, about 150 nucleotides away from the closest canonical splice site. By sequencing a number of independent cDNA clones covering exons 14 to 17, we demonstrated that this mutation reduced splicing efficiency but did not completely abrogate the production of the normal transcript. Prompted by this finding, we sequenced the same genomic region in 33 patients from our unresolved ARO cohort and found three additional novel single nucleotide changes in a similar location and with a predicted disruptive effect on splicing, further confirmed in one of them at the transcript level. Overall, we identified an intronic region in TCIRG1 that seems to be particularly prone to splicing mutations, allowing the production of a small amount of protein sufficient to reduce the severity of the phenotype usually associated with TCIRG1 defects. On this basis, we would recommend including TCIRG1 not only in the molecular work-up of severe infantile osteopetrosis but also in intermediate cases and carefully evaluating the possible effects of intronic changes. © 2015 American Society for Bone and Mineral Research.