Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as “splicing mutations,” but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002–1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5′- and 3′-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002–1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75

3D kinematics through the X-shaped Milky Way bulge

Context. It has recently been discovered that the Galactic bulge is X-shaped, with the two southern arms of the X both crossing the lines of sight at l = 0 and | b| > 4, hence producing a double red clump in the bulge color magnitude diagram. Dynamical models predict the formation of X-shaped bulges as extreme cases of boxy-peanut bulges. However, since X-shaped bulges were known to be present only in external galaxies, models have never been compared to 3D kinematical data for individual stars. Aims. We study the orbital motion of Galactic bulge stars in the two arms (overdensities) of the X in the southern hemisphere. The goal is to provide observational constraints to bulge formation models that predict the formation of X-shapes through bar dynamical instabilities. Methods. Radial velocities have been obtained for a sample of 454 bulge giants, roughly equally distributed between the bright and the faint red clump, in a field at (l,b) = (0, −6). Proper motions were derived for all red clump stars in the same field by combining images from two epochs, which were obtained 11 years apart, with WFI at the 2.2 m at La Silla. The observed field contains the globular cluster NGC 6558, whose member stars were used to assess the accuracy of the proper motion measurement. At the same time, as a by-product, we provide the first proper motion measurement of NGC 6558. The proper motions for the spectroscopic subsample are analyzed for a subsample of 352 stars, taking into account the radial velocities and metallicities measured from near-infrared calcium triplet lines. Results. The radial velocity distribution of stars in the bright red clump, which traces the closer overdensity of bulge stars, shows an excess of stars moving towards the Sun. Similarly, an excess of stars receding from the Sun is seen in the far overdensity, which is traced by faint red clump stars. This is explained by the presence of stars on elongated orbits, which are most likely streaming along the arms of the X-shaped bulge. Proper motions for these stars are consistent with qualitative predictions of dynamical models of peanut-shaped bulges. Surprisingly, stars on elongated orbits have preferentially metal-poor (subsolar) metallicities, while the metal rich ones, in both overdensities, are preferentially found in more axisymmetric orbits. The observed proper motion of NGC 6558 has been measured as (μlcos   (b),μb) = (0.30   ±   0.14, −0.43 ± 0.13), with a velocity dispersion of (σlcos(b),σb) = (1.8,1.7) mas/yr. This is the first proper motion measurement for this cluster

Role of genetic investigation in the diagnosis of short stature in a cohort of Italian children

Background: Short stature (SS) is defined as height more than 2 standard deviations below the mean for age and sex. Hypothyroidism, celiac disease, growth hormone deficiency, hormonal abnormalities, and genetic conditions are among its causes. A wide range of conditions often due to largely unknown genetic variants can elude conventional diagnostic workup. Aim: We used next-generation sequencing (NGS) to better understand the etiology of SS in a cohort of Italian children. Patients and methods: The study sample was 125 children with SS of unknown origin referred to our Institute between 2015 and 2021. All had undergone complete auxological and hormonal investigations to exclude common causes of SS. Genetic analysis was performed using a NGS panel of 104 genes. Clinical data were reviewed to clarify the pathogenicity of the variants detected. Results: In this cohort, 43 potentially causing variants were identified in 38 children. A syndromic genetic condition was diagnosed in 7: Noonan syndrome in 3, Leri-Weill syndrome in 3, and hypochondroplasia in 1. Moreover, 8 benign variants and other 37 like benign variants were found. In 88 children, 179 variants of uncertain significance (VUS) were identified. No variant was found in 16 children. Conclusion: Genetic analysis is a useful tool in the diagnostic workup of patients with SS, in adapting management and treatment, and in identifying syndromes with mild atypical clinical features. The role of VUS should not be underestimated, particularly when multiple VUS with possible mutual worsening effects are present in the same child

Germline mosaicism in achondroplasia detected in sperm DNA of the father of three affected sibs

We describe a sib recurrence for achondroplasia with parents of average stature. The three sibs shared the paternal allele and all carried the same causal mutation in the fibroblast growth factor receptor 3 gene (FGFR3): G > A nt1138 (Gly380Arg). We were able to identify this mutation on sperm DNA confirming paternal germinal mosaicism. Our family shows that a more precise definition of the recurrence risk is feasible using this approach, based on a single DNA test, which could be offered in selected cases

Bilateral carcinoma in situ of the testis and cystic fibrosis transmembrane conductance regulator (CFTR) mutation in an azoospermic patient with late-onset 21beta-hydroxylase deficiency.

Testicular cancer can impair spermatogenesis. In addition, chemotherapy or radiotherapy used for its treatment further damage testicular function mainly affecting highly proliferating germ cells. The multifaceted etiology of male infertility includes, among others, alterations of male reproductive tract differentiation such as monolateral or bilateral congenital absence of vas deferens and perturbations in adrenal steroid synthesis on a genetic basis such as 21beta-hydroxylase deficiency. Herein, we report the case of a male patient with primary infertility, probably related to a combination of genetic and acquired factors with different expressions over tim

Duplications upstream and downstream of SHOX identified as novel causes of Leri-Weill dyschondrosteosis or idiopathic short stature.

Leri-Weill dyschondrosteosis is a pseudoautosomal dominantly-inherited skeletal dysplasia ascribed to haploinsufficiency of the SHOX gene caused by deletions, point mutations, or partial duplications of the gene, or to heterozygous deletions upstream or downstream of the intact SHOX gene involving conserved non-coding cis-regulatory DNA elements that show enhancer activity. Recently, two SHOX conserved non-coding element duplications, one upstream and one downstream, were reported in patients referred with idiopathic short stature. To further evaluate the role of these duplications in SHOX-related disorders, we describe seven patients (five with Leri-Weill dyschondrosteosis and two with short stature) all of whom have duplications of part of the upstream or downstream conserved non-coding element regions, identified by multiplex ligation-dependent probe amplification. In addition, we show data from 32 patients with an apparently identical downstream duplication that includes a proposed putative regulatory element (identified by multiplex ligation-dependent probe amplification or array comparative genome hybridization), which results in a variable phenotype from normal to mild Leri-Weill dyschondrosteosis. These additional data provide further evidence that duplications of upstream and downstream long range cis-regulatory DNA elements can result in a SHOX-related phenotype

Performance Assessment of Thermal Infrared Cameras of Different Resolutions to Estimate Tree Water Status from Two Cherry Cultivars: An Alternative to Midday Stem Water Potential and Stomatal Conductance

The midday stem water potential (Ψs) and stomatal conductance (gs) have been traditionally used to monitor the water status of cherry trees (Prunus avium L.). Due to the complexity of direct measurement, the use of infrared thermography has been proposed as an alternative. This study compares Ψs and gs against crop water stress indexes (CWSI) calculated from thermal infrared (TIR) data from high-resolution (HR) and low-resolution (LR) cameras for two cherry tree cultivars: 'Regina' and 'Sweetheart'. For this purpose, a water stress-recovery cycle experiment was carried out at the post-harvest period in a commercial drip-irrigated cherry tree orchard under three irrigation treatments based on Ψs levels. The water status of trees was measured weekly using Ψs, gs, and compared to CWSIs, computed from both thermal cameras. Results showed that the accuracy in the estimation of CWSIs was not statistically significant when comparing both cameras for the representation of Ψs and gs in both cultivars. The performance of all evaluated physiological indicators presented similar trends for both cultivars, and the averaged differences between CWSI's from both cameras were 11 ± 0.27%. However, these CWSI's were not able to detect differences among irrigation treatments as compared to Ψs and gs