[Analysis of GNAS gene variant in a Chinese pedigree affected with pseudohypoparathyroidism].

OBJECTIVE: To explore the genetic etiology of a Chinese pedigree affected with pseudohypoparathyroidism. METHODS: Peripheral blood samples of the proband and his parents were collected and subjected to trio-whole exome sequencing (trio-WES). Candidate variants were verified among the pedigree and 50 randomly selected healthy individuals through analysis of restriction fragment length polymorphism. Short tandem repeat (STR) linkage analysis was used to verify the parental origin of the pathogenic variants. RESULTS: Trio-WES and Sanger sequencing showed that the proband and his mother had both harbored a c.121C>G (p.His41Asp) variant of the GNAS gene, which was not found in other family members and the 50 healthy controls. The variant was not found in international databases. Based on guidelines from the American College of Medical Genetics and Genomics, the variant was predicted to be likely pathogenic. CONCLUSION: The novel c.121C>G variant of the GNAS gene probably underlay the disease in this pedigree. Above finding has enriched the spectrum of GNAS gene variants.

[Effects of Acrolein on the Proliferation of and Per1 Gene Expression in Pulmonary Epithelial Cells].

OBJECTIVE: To investigate the effect of acrolein on the proliferation of pulmonary epithelial cells and its possible mechanism. METHODS: Two strains of pulmonary epithelial cells, A549 cells and MLE15 cells, were used as in vitro models of pulmonary epithelial cell, and were treated with 80 µmol/L acrolein or phosphate buffer saline (PBS) as the control. The proliferation of pulmonary epithelial cells were determined with CCK-8 kit after cell culturing resumed for 12 h, 24 h, 36 h and 48 h post acrolein treatment, and the expression of period circadian regulator gene 1 ( Per1) was examined using Western blot test 24 h after acrolein treatment. In addition, after acrolein treatment, the cells were restored with transforming growth factor-ß (TGF-ß) added in the medium, and the cell proliferation and the expression of Per1 protein were also examined. RESULTS: The proliferation of A549 cells and MLE15 cells decreased significantly after being treated with 80 µmol/L acrolein for 30 min, and the expression of Per1 protein was also downregulated significantly ( P<0.05). The addition of TGF-ß after acrolein treatment did not significantly change the reduction in cell proliferation caused by acrolein, but the expression of Per1 protein in pulmonary epithelial cells was significantly higher than that in cells restored without TGF-ß ( P<0.05). CONCLUSION: Acrolein treatment resulted in the decreased proliferation of pulmonary epithelial cells and the Per1 expression in pulmonary epithelial cells. Although TGF-ß addition did not reverse the reduction of cell proliferation after acrolein treatment, the Per1 expression levels were recovered to a certain extent compared to that in cells restored in medium without TGF-ß after acrolein treatment.

Structural and functional conservation of key domains in InsP3 and ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca(2+) channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP(3)R gating is initiated by InsP(3) binding to the InsP(3)-binding core (IBC, residues 224-604 of InsP(3)R1) and it requires the suppressor domain (SD, residues 1-223 of InsP(3)R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP(3)R1 with (3.6 Å) and without (3.0 Å) InsP(3) bound. The arrangement of the three NT domains, SD, IBC-ß and IBC-α, identifies two discrete interfaces (α and ß) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP(3)R and of the ABC domains docked into RyR are remarkably similar. The importance of the α-interface for activation of InsP(3)R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP(3) causes partial closure of the clam-like IBC, disrupting the ß-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for InsP(3)R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP(3)R, and an InsP(3)R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP(3) and blocked by ryanodine. Activation mechanisms are conserved between InsP(3)R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-ß or B domain), to gate the pore.

CaBP1, a neuronal Ca2+ sensor protein, inhibits inositol trisphosphate receptors by clamping intersubunit interactions.

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca(2+) channels, including inositol 1,4,5-trisphosphate receptors (InsP3Rs). CaBP1 alone does not affect InsP3R activity, but it inhibits InsP3-evoked Ca(2+) release by slowing the rate of InsP3R opening. The inhibition is enhanced by Ca(2+) binding to both the InsP3R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1-604) of InsP3R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca(2+) binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the ß-domain of the InsP3-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP3R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP3R pore. InsP3 activates InsP3Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a "hot-spot" loop in the suppressor domain (residues 1-223) and the InsP3-binding core ß-domain. Targeted cross-linking of residues that contribute to this interface show that InsP3 attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP3R activity by restricting the intersubunit movements that initiate gating.

Structural insights into activation of the retinal L-type Ca²âº channel (Cav1.4) by Ca²âº-binding protein 4 (CaBP4).

CaBP4 modulates Ca(2+)-dependent activity of L-type voltage-gated Ca(2+) channels (Cav1.4) in retinal photoreceptor cells. Mg(2+) binds to the first and third EF-hands (EF1 and EF3), and Ca(2+) binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg(2+)-bound and Ca(2+)-bound states and model the CaBP4 structural interaction with Cav1.4. CaBP4 contains an unstructured N-terminal region (residues 1-99) and four EF-hands in two separate lobes. The N-lobe consists of EF1 and EF2 in a closed conformation with either Mg(2+) or Ca(2+) bound at EF1. The C-lobe binds Ca(2+) at EF3 and EF4 and exhibits a Ca(2+)-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca(2+)-bound CaBP4 (Phe(137), Glu(168), Leu(207), Phe(214), Met(251), Phe(264), and Leu(268)) make contacts with the IQ motif in Cav1.4, and the Cav1.4 mutant Y1595E strongly impairs binding to CaBP4. We conclude that CaBP4 forms a collapsed structure around the IQ motif in Cav1.4 that we suggest may promote channel activation by disrupting an interaction between IQ and the inhibitor of Ca(2+)-dependent inactivation domain.

Association of DNMT1 Gene Polymorphisms with Congenital Heart Disease in Child Patients.

To reveal the association between DNMT1 polymorphisms and congenital heart disease (CHD) in child patients, a total of 224 CHD child patients as well as 199 healthy individuals were enrolled in the present study. The DNA was extracted from whole blood, and four SNPs including rs16999593, rs2228612, rs2288349 and rs10420321 were selected for the gene polymorphism investigation via ligase detection reaction (LDR) assay. Odds ratios (ORs) and 95 % confidence intervals (95 % CIs) were used to assess the strength of the association. rs16999593 was associated with the CHD under the heterozygous (CT vs TT: OR 0.62; 95 % CI 0.41-0.95; p = 0.03), dominant (CT + CC vs TT: OR 0.63; 95 % CI 0.42-0.95; p = 0.03), and allele models (C vs T: OR 0.07; 95 % CI 0.50-1.00; p = 0.05). rs2228612 was related with the CHD under the heterozygous (AG vs AA: OR 0.42; 95 % CI 0.27-0.65; p = 0.0001), homozygous (GG vs AA: OR 0.43; 95 % CI 0.240-0.77; p = 0.004), dominant (AG + GG vs AA: OR 0.42; 95 % CI 0.28-0.64; p < 0.0001), and allele models (G vs A: OR 0.62; 95 % CI 0.47-0.82; p = 0.0007). rs10420321 correlated with the CHD only under the recessive model (GG vs AG + AA: OR 0.61; 95 % CI 0.37-1.01, p = 0.05). However, no significant association between the rs2288349 polymorphisms and the risk of CHD was observed (p > 0.05). DNMT1 polymorphisms might contribute to the risk of CHD, especially rs16999593 and rs2228612.

Double electron-electron resonance probes Ca²âº-induced conformational changes and dimerization of recoverin.

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²âº-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²âº-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²âº-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²âº-free recoverin and 3.7 ± 0.1 nm for Ca²âº-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²âº-bound recoverin may represent an intermolecular distance between C39 and N120. ¹5N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²âº-bound recoverin forms a dimer at protein concentrations above 100 µM, whereas Ca²âº-free recoverin is monomeric. We propose that Ca²âº-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²âº-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²âº-induced dimerization is functionally important but not well understood.

Application of "Internet+" cognitive-behavioral intervention in caregivers of children with congenital heart disease undergoing elective surgery during the COVID-19 pandemic.

OBJECTIVE: To explore the effects of cognitive and behavioral interventions for caregivers of children undergoing interventional surgery for congenital heart disease (CHD) during COVID-19. METHODS: A prospective study was conducted on 140 children with CHD who were hospitalized in the Department of Cardiology in a children's hospital from March 2020 to March 2022. The children were randomly divided into an intervention group and a control group, with 70 cases in each group. In the control group, caregivers gave routine care, and in the intervention group, "Internet+" cognitive and behavioral interventions were given. The psychological status of caregivers before and after intervention, day care ability on the operation day, readiness for hospital discharge of the caregivers, sleep quality, and postoperative complications of the children, the medication compliance, review compliance and satisfaction were compared between the two groups. RESULTS: During the COVID-19 pandemic, the anxiety and depression scores of caregivers in the intervention group were significantly lower than those in the control group (P<0.05), and the caregiving ability and readiness for hospital discharge of the caregivers in the intervention group were better than those in the control group (P<0.05). The sleep quality of children in the intervention group was significantly better than that in the control group during the first week after operation (P<0.05). Postoperative complications were significantly fewer in the intervention group than in the control group (χ 2=24.433, P<0.001). The medication compliance, review compliance, and satisfaction were higher in the intervention group than in the control group (P<0.05). CONCLUSION: During the pandemic period of COVID-19, "Internet+" cognitive and behavioral intervention has a good effect and should be promoted in clinical practice.

Structural analysis of Mg2+ and Ca2+ binding, myristoylation, and dimerization of the neuronal calcium sensor and visinin-like protein 1 (VILIP-1).

Visinin-like protein 1 (VILIP-1) belongs to the neuronal calcium sensor family of Ca(2+)-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca(2+) and Mg(2+) binding, characterize metal-induced conformational changes, and determine structural effects of myristoylation and dimerization. Mg(2+) binds functionally to VILIP-1 at EF3 (ΔH = +1.8 kcal/mol and K(D) = 20 µM). Unmyristoylated VILIP-1 binds two Ca(2+) sequentially at EF2 and EF3 (K(EF3) = 0.1 µM and K(EF2) = 1-4 µM), whereas myristoylated VILIP-1 binds two Ca(2+) with lower affinity (K(D) = 1.2 µM) and positive cooperativity (Hill slope = 1.5). NMR assignments and structural analysis indicate that Ca(2+)-free VILIP-1 contains a sequestered myristoyl group like that of recoverin. NMR resonances of the attached myristate exhibit Ca(2+)-dependent chemical shifts and NOE patterns consistent with Ca(2+)-induced extrusion of the myristate. VILIP-1 forms a dimer in solution independent of Ca(2+) and myristoylation. The dimerization site is composed of residues in EF4 and the loop region between EF3 and EF4, confirmed by mutagenesis. We present the structure of the VILIP-1 dimer and a Ca(2+)-myristoyl switch to provide structural insights into Ca(2+)-induced trafficking of nicotinic acetylcholine receptors.

[Mutation analysis of pathogenic genes in a Henan family affected with congenital stationary night blindness].

OBJECTIVE: To detect genetic mutations associated with autosomal dominant congenital stationary night blindness (ADCSNB) in a family from Henan province. METHODS: Genomic DNA was extracted from peripheral blood samples of 14 family members. Based on 3 genes reported previously, PCR primers were designed and corresponding exons containing the mutation sites were amplified with PCR. PCR products were purified and directly sequenced. RESULTS: A c.281C>T heterozygous missense mutation was detected in RHO gene in all of the patients. This mutation can cause a change of the protein structure (p.Thr94Ile). The same mutation was not detected in normal individuals from the family and 50 normal controls. CONCLUSION: A c.281C>T mutation in RHO gene is responsible for the onset of ADCSNB in this Chinese family and results in symptoms of night blindness.

[Mutation analysis of the pathogenic gene in a Chinese family with Brachydactyly type B1].

We identified and characterized a Chinese family with autosomal dominant Brachydactyly type B1 (BDB1). Linkage analysis revealed that the disease gene of the Chinese BDB1 family was linked to ROR2 locus. Mutational hot spot of ROR2 gene was amplified by polymerase chain reaction (PCR) and sequenced directly. A c.2265C>A heterozygous mutation was detected in all of the patients. This mutation led to the change of p.Y755X in protein level and a truncated ROR2 protein losing integrant domains was generated. The mutation was detected in all the patients, but not in all the normal individuals of this family and 50 normal controls. This paper for the first time reported a c.2265C>A mutation in ROR2 gene of a family with BDB1 in China, which enriches ROR2 gene mutation spectrum in Chinese with BDB1.

[The mutation spectrum of phenylalanine hydroxylase gene in patients with phenylketonuria in Henan province].

OBJECTIVE: To investigate the characteristics of the phenylalanine hydroxylase (PAH) gene mutations in patients with phenylketonuria (PKU) in Henan province, China, in order for providing basic information for clinical genetic counseling and prenatal diagnosis. METHODS: All the exons and partial flanking introns of the PAH gene were detected by polymerase chain reaction (PCR) and bi-directional sequencing in 34 patients with PKU from Henan province. RESULTS: A total of 23 different disease-causing mutations were identified which corresponded to 92.65% (63/68) of the PAH alleles, including 12 missense mutations, 4 nonsense mutations, 4 splicing junction mutations, and 3 deletion mutations. Among them, A156P and P69_S70delinsP(delCTT) were novel mutations; IVS2+ 5G to C, G332E, IVS10-14C to G and L367 to Wfs were reported in Chinese population for the first time according to the PAH database (www.pahdb.mcgill.ca). Among all the 13 exons, exon 7 harbored the most type of mutations, exon 11 and exon 5 the second. The most common mutations included R243Q (17.65%, 12/68), V399V (11.76%, 8/68), IVS4-1G to A (8.82%, 6/68), R400T(7.35%, 5/68), Y166X(5.88%,4/68) and G247R(5.88%, 4/68). In addition, 9 other gene variations were found in this study. CONCLUSION: The mutation spectrum and frequency of the PAH gene of patients with phenylketonuria in Henan province were slightly different from those from other parts of China.

Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3.

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 µM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.

¹H, ¹³C, and ¹5N chemical shift assignments of neuronal calcium sensor protein, hippocalcin.

Hippocalcin, a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, serves as an important calcium sensor for the slow afterhyperpolarizing (sAHP) current in the hippocampus, which underlies some forms of learning and memory. Hippocalcin is also a calcium sensor for hippocampal long-term depression (LTD) and genetically linked to neurodegenerative diseases. We report NMR chemical shift assignments of Ca(2+)-free hippocalcin (BMRB no. 18627).

¹H, ¹5N, and ¹³C chemical shift assignments of murine calcium-binding protein 4.

Calcium-binding protein 4 (CaBP4) regulates voltage-gated Ca(2+) channels in retinal rod cells and specific mutations within CaBP4 are associated with congenital stationary night blindness type 2. We report complete NMR chemical shift assignments of the Ca(2+)-saturated form of CaBP4 with Ca(2+) bound at EF1, EF3 and EF4 (BMRB no. 18877).

Variations of CITED2 are associated with congenital heart disease (CHD) in Chinese population.

CITED2 was identified as a cardiac transcription factor which is essential to the heart development. Cited2-deficient mice showed cardiac malformations, adrenal agenesis and neural crest defects. To explore the potential impact of mutations in CITED2 on congenital heart disease (CHD) in humans, we screened the coding region of CITED2 in a total of 700 Chinese people with congenital heart disease and 250 healthy individuals as controls. We found five potential disease-causing mutations, p.P140S, p.S183L, p.S196G, p.Ser161delAGC and p. Ser192_Gly193delAGCGGC. Two mammalian two-hybrid assays showed that the last four mutations significantly affected the interaction between p300CH1 and CITED2 or HIF1A. Further studies showed that four CITED2 mutations recovered the promoter activity of VEGF by decreasing its competitiveness with HIF1A for binding to p300CH1 and three mutations decreased the consociation of TFAP2C and CITED2 in the transactivation of PITX2C. Both VEGF and PITX2C play very important roles in cardiac development. In conclusion, we demonstrated that CITED2 has a potential causative impact on congenital heart disease.

Homeobox C9 is not potentially related to congenital heart disease in Chinese patients.

BACKGROUND: Congenital heart disease (CHD) is one of the most common human birth defects. The etiology and pathogenesis of CHD are complex and involve several genes as well as multiple changes in signaling pathways. The aim of this study was to identify potential pathological mutations in the Homeobox C9 (Hoxc9) gene in 350 Chinese children with CHD to further understand the etiology of CHD. METHOD: Sequence analysis of the Hoxc9 gene in 350 nonsyndromic patients with CHD Result: We did not identify any nonsynonymous variants in the coding regions of Hoxc9 in the patients with CHD. We found one synonymous variant c.C564T (p. his188his) in one ventricular septal defect patient. We also identified four previously reported polymorphisms (rs56368105, rs12817092, rs34079606, and rs2241820) in CHD. CONCLUSIONS: We did not find any diagnostic alterations in the coding regions of Hoxc9 among the patients with CHD. Nevertheless, to our knowledge, this is the first study of Hoxc9 in nonsyndromic CHD and has expanded our overall knowledge of the etiology of this disease.

Nuclear magnetic resonance structure of calcium-binding protein 1 in a Ca(2+) -bound closed state: implications for target recognition.

Calcium-binding protein 1 (CaBP1), a neuron-specific member of the calmodulin (CaM) superfamily, regulates the Ca(2+) -dependent activity of inositol 1,4,5-triphosphate receptors (InsP3Rs) and various voltage-gated Ca(2+) channels. Here, we present the NMR structure of full-length CaBP1 with Ca(2+) bound at the first, third, and fourth EF-hands. A total of 1250 nuclear Overhauser effect distance measurements and 70 residual dipolar coupling restraints define the overall main chain structure with a root-mean-squared deviation of 0.54 Å (N-domain) and 0.48 Å (C-domain). The first 18 residues from the N-terminus in CaBP1 (located upstream of the first EF-hand) are structurally disordered and solvent exposed. The Ca(2+) -saturated CaBP1 structure contains two independent domains separated by a flexible central linker similar to that in calmodulin and troponin C. The N-domain structure of CaBP1 contains two EF-hands (EF1 and EF2), both in a closed conformation [interhelical angles = 129° (EF1) and 142° (EF2)]. The C-domain contains EF3 and EF4 in the familiar Ca(2+) -bound open conformation [interhelical angles = 105° (EF3) and 91° (EF4)]. Surprisingly, the N-domain adopts the same closed conformation in the presence or absence of Ca(2+) bound at EF1. The Ca(2+) -bound closed conformation of EF1 is reminiscent of Ca(2+) -bound EF-hands in a closed conformation found in cardiac troponin C and calpain. We propose that the Ca(2+) -bound closed conformation of EF1 in CaBP1 might undergo an induced-fit opening only in the presence of a specific target protein, and thus may help explain the highly specialized target binding by CaBP1.

1H, 15N, and 13C chemical shift assignments of calcium-binding protein 1 with Ca2+ bound at EF1, EF3 and EF4.

Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) and a variety of voltage-gated Ca(2+) channels in the brain. We report complete NMR chemical shift assignments of the Ca(2+)-saturated form of CaBP1 with Ca(2+) bound at EF1, EF3 and EF4 (residues 1-167, BMRB no. 16862).