Evaluation of genetic counseling among cystic fibrosis carriers, Michigan Newborn Screening

Objective A quality improvement (QI) strategy to improve the rate of genetic counseling (GC) services was initiated in cystic fibrosis (CF) care Center E in 2010. This statewide study was conducted to determine: (1) GC rates before and after implementation of the QI strategy at Center E; (2) characteristics associated with not receiving GC; and (3) topic areas addressed during GC. Methods The retrospective study included 1,097 CF carriers born from 2008 to 2011 identified through Michigan's Newborn Screening Program. Rate of GC services was determined for Center E and the other four CF centers before and after the QI change. Bivariate and multivariable logistic regression was used to determine associations between select characteristics and not receiving GC. Topic areas discussed during GC sessions were assessed using frequency tables. Results Rate of GC services in Center E increased from 23% in 2008–2010 to 91% in 2011, while at the other centers approximately 92% received GC services across those years. In 2008–2010, being seen at Center E and black race were significantly associated with increased likelihood of not receiving GC services in adjusted analyses. In 2011, neither characteristic was associated with receipt of GC. Of 16 target topic areas, all were discussed in 85% of GC sessions. Conclusions Implementing a QI strategy of providing sweat test results at the GC appointment within Center E resulted in more CF carriers receiving comprehensive GC services. Center‐specific procedure differences should be assessed to increase rate of GC services following a positive CF newborn screen. Pediatr Pulmonol. 2013; 48:123–129. © 2012 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96254/1/22703_ftp.pd

<i>slc25a25b</i> is required for left–right organ patterning in zebrafish.

<p>(A,B) As a measure for left–right asymmetry, heart looping of <i>slc25a25b</i>-morphant fish was visualized by in situ hybridization for <i>cmlc2</i>. (C) Knockdown of <i>slc25a25b</i> caused randomization of heart looping (<i>slc25a25b</i>^<i>MO</i>; *<i>P</i> = 1.1 × 10⁻³⁴). This phenotype was rescued by injection (+) of <i>slc25a25b</i> mRNA in a concentration-dependent fashion (<i>+slc25a25b</i>). (D-F) <i>southpaw</i> (<i>nodal</i>) expression in 15-somite stage <i>slc25a25b</i>-morphant zebrafish embryos was visualized by in situ hybridization. (G) In contrast to control fish, in which <i>southpaw</i> expression was largely restricted to the left side, <i>slc25a25b</i>-morphant fish showed randomized <i>southpaw</i> expression (*<i>P</i> = 0.00008). Numbers of embryos are indicated above bars. L = left; S = symmetric; R = right. This denomination for heart looping is equivalent to wt <i>d-loop</i>, symmetric <i>no-loop</i>, and reversed, sinistral <i>s-loop</i> [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005651#pbio.2005651.ref033" target="_blank">33</a>]. For numerical values, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005651#pbio.2005651.s014" target="_blank">S1 Data</a>. <i>cmlc2</i>, <i>cardiac myosin light chain 2</i>; hpf, hours post fertilization; wt, wild type.</p

SCaMC is a Ca²⁺-regulated protein required for sperm storage in <i>Drosophila</i>.

<p>(A) <i>D</i>. <i>melanogaster</i> TRPP2 mutants (<i>amo</i>^<i>1</i>) were male sterile (*<i>P</i> = 9 × 10⁻¹¹). (B) Homozygous <i>Z3-2147</i> males and males with <i>Z3-2147</i> over a third-chromosomal deficiency deleting <i>CG32103</i>, <i>Df(3L)BSC380</i>, were infertile (both *<i>P</i> = 0.003). <i>Z3-2147/+</i> heterozygous males showed normal fertility. One copy of the <i>CG32103</i> cDNA fully rescued the fertility defect in <i>Z3-2147</i> homozygous males (<i>CG32103/+;Z3-2147</i>). (C) Sequencing chromatogram of <i>SCaMC</i>^<i>1</i> (<i>Z3-2147</i>) flies. Mutant flies presented with a point mutation on the third chromosome at position 3L:12,405,022, causing a change of thymine to adenine. (D) This missense mutation translates into replacement of basic arginine (R) 308 to nonpolar tryptophan (W) in the evolutionarily highly conserved first mitochondrial carrier domain of SCaMC (CG32103). (E) Mutations in the EF-hand domains of SCaMC (CG32103) impaired its function. <i>SCaMC</i> variants were tested in <i>SCaMC</i>^<i>1</i> (CG32103^R308W) mutant background. While transgenic rescue with <i>SCaMC</i> wt cDNA (<i>+SCaMC</i>) behaved like wt males, transgenes lacking the N-terminal EF-hands (+<i>SCaMC</i>^{<i>Δ1–268</i>}) or transgenes comprising missense mutations inactivating the Ca²⁺-binding EF-hand domains (+<i>SCaMC</i>^<i>EF</i>) barely rescued <i>SCaMC</i>^<i>1</i> male fertility (both *<i>P</i> = 0.0009; compared to wt rescue). (F) Human SLC25A25 cDNA rescued infertility in <i>SCaMC</i>^<i>1</i> homozygous males (<i>SLC25A25/+; SCaMC</i>^<i>1</i>) (*<i>P</i> = 0.0001). For numerical values, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005651#pbio.2005651.s014" target="_blank">S1 Data</a>. SLC25A25, solute carrier 25 A 25; TRPP2, transient receptor potential channel polycystin-2; wt, wild-type.</p

Metabolic profiles of TRPP2- and SLC25A25-deficient cells.

<p>(A) The ratio of metabolites in <i>Slc25a25</i>-deficient and wt mIMCD3 cells are plotted against their <i>P</i> value (each dot represents 1 metabolite). The analysis showed 42 significantly changed metabolites in <i>Slc25a25</i>-deficient cells (blue dots; <i>n</i> = 6, *<i>P</i> ≤ 0.00019). (I.) decreased, not significant; (II.) increased, not significant; (III.) significantly increased; and (IV.) significantly decreased. (B) Statistical evaluation of metabolite ratios comparing mIMCD3 <i>Pkd2</i>^{<i>−/−</i>} and wt cells. The analysis showed 55 significantly changed metabolites in <i>Pkd2</i>-deficient cells (blue dots; <i>n</i> = 6, *<i>P</i> ≤ 0.00019). (C) Ratios of metabolites of SLC25A25-deficient and TRPP2-deficient cells (<i>Pkd2</i>^{<i>−/−</i>}) relative to wt cells showed concordant changes of several metabolites. Four metabolites were significantly increased (III.) and 7 significantly decreased (I.) in both knockout cell lines (blue dots; <i>n</i> = 6, *<i>P</i> ≤ 0.00019). (D) Relative abundance of metabolites with concordant changes in TRPP2- and SLC25A25-deficient cells compared to wt cells (metabolites from quadrant I. and III. in panel A). (E) Cellular ATP content was decreased in SLC25A25- and TRPP2-deficient cells (depicted as relative change compared to wt cells; <i>n</i> = 6, *<i>P</i> ≤ 0.01). For numerical values, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005651#pbio.2005651.s015" target="_blank">S2 Data</a>. SLC25A25, solute carrier 25 A 25; TRPP2, transient receptor potential channel polycystin-2; wt, wild type.</p

SLC25A25 is a Ca²⁺-regulated Mg-ATP transporter.

<p>(A) Purified SLC25A25 (5 μg) was separated by SDS-PAGE and visualized by Coomassie staining. (B) Thermostability profile (top) and its corresponding first derivative (bottom) of SLC25A25. Assays were performed in triplicate with the addition CaCl₂ (orange line) or without (blue line). (C) Uptake assays of [¹⁴C]-labeled Mg-ATP into proteoliposomes with the addition CaCl₂ (orange line) and without (blue line). The error bars represent the standard deviation of 4 replicates, and the uptake curves were fitted with a one-phase association curve (r-squared > 0.99). For numerical values, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005651#pbio.2005651.s014" target="_blank">S1 Data</a>. AU, arbitrary unit; SLC25A25, solute carrier 25 A 25.</p

TRPP2 and SLC25A25 act in a common signaling cascade required for vertebrate left–right patterning.

<p>(A) Conceptually, our data imply a function of SLC25A25 (Slc25a25b) between flow-mediated asymmetric activation (green) of TRPP2-dependent Ca²⁺ signaling in KV and Nodal-dependent asymmetric gene expression in the lateral plate mesoderm (red). (B) Randomized left–right asymmetry in <i>pkd2</i>-morphant zebrafish (<i>pkd2</i>^<i>MO</i>; *<i>P</i> = 6 × 10⁻²¹) was partially rescued by expression of <i>slc25a25b</i> mRNA (*<i>P</i> = 4 × 10⁻⁶). (C) Randomized left–right asymmetry in <i>pkd2-</i>null (<i>pkd2</i>^{<i>−/−</i>}) zebrafish (*<i>P</i> = 9 × 10⁻¹⁵) was not rescued by expression of <i>slc25a25b</i> mRNA (*<i>P</i> = 0.27). Numbers of embryos are indicated above bars. L = left; S = symmetric; R = right. (D) Schematic of functional coupling of TRPP2 and SLC25A25. For numerical values, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005651#pbio.2005651.s014" target="_blank">S1 Data</a>. KV, Kupffer’s vesicle; SLC25A25, solute carrier 25 A 25; TRPP2, transient receptor potential channel polycystin-2.</p

Schematic of the workflow to identify components of the TRPP2 signaling cascade.

<p>A forward genetic screen in <i>D</i>. <i>melanogaster</i> identified SCaMC as a potential downstream target of TRPP2. The evolutionary conservation of the SCaMC vertebrate homolog SLC25A25 in TRPP2-dependent signaling was validated in zebrafish. Genome editing, transport assays, and metabolomics were used to investigate the molecular function of SLC25A25 in vitro, suggesting a mechanistic link between cilia and mitochondria. SLC25A25, solute carrier 25 A 25; TRPP2, transient receptor potential channel polycystin-2.</p

TRPP2 and SLC25A25 form a signaling microdomain.

<p>(A) Indirect immunofluorescence of SLC25A25^V5 showed colocalization with the mitochondrial marker pDsRed2-Mito (scale bar = 10 μm). (B) Transmission electron microscopy of a ciliated cell from KV (arrowhead: cilium; arrows: mitochondria; scale bar = 200 nm). Representative of 3 images with a similar pattern. (C) Indirect immunofluorescence of SLC25A25^V5 (magenta) and TRPP2 (green) showed tight spatial coupling of both proteins at ER–mitochondria junctions in epithelial cells using STED microscopy (scale bar = 5 μm; inset = 1 μm). The inset shows 3D rendering of a magnification depicting close contacts between the ER and mitochondria (scale bar = 1 μm). ER, endoplasmic reticulum; KV, Kupffer’s vesicle; SLC25A25, solute carrier 25 A 25; SLC25A25^V5, V5-tagged SLC25A25; STED, stimulated emission depletion; TRPP2, transient receptor potential channel polycystin-2.</p