A Novel Missense Mutation of GATA4 in a Chinese Family with Congenital Heart Disease

<div><p>Background</p><p>Congenital heart disease (CHD) is the most prevalent type of birth defect in human, with high morbidity in infant. Several genes essential for heart development have been identified. GATA4 is a pivotal transcription factor that can regulate the cardiac development. Many GATA4 mutations have been identified in patients with different types of CHD.</p><p>Aims</p><p>In this study, the NKX2-5, HAND1 and GATA4 coding regions were sequenced in a family spanning three generations in which seven patients had CHD. Disease-causing potential variation in this family was evaluated by bioinformatics programs and the transcriptional activity of mutant protein was analyzed by the dual luciferase reporter assay.</p><p>Results</p><p>A novel GATA4 mutation, c.C931T (p.R311W), was identified and co-segregated with the affected patients in this family. The bioinformatics programs predicted this heterozygous mutation to be deleterious and the cross-species alignment of GATA4 sequences showed that the mutation occurred within a highly conserved amino acid. Even though it resided in the nuclear localization signal domain, the mutant protein didn’t alter its intracellular distribution. Nevertheless, further luciferase reporter assay demonstrated that the p.R311W mutation reduced the ability of GATA4 to activate its downstream target gene.</p><p>Conclusions</p><p>Our study identified a novel mutation in GATA4 that likely contributed to the CHD in this family. This finding expanded the spectrum of GATA4 mutations and underscored the pathogenic correlation between GATA4 mutations and CHD.</p></div

Distribution of the identified GATA4 mutation and multiple sequence alignment of GATA4 protein.

<p>(A) Sequencing results of the GATA4 mutation. The arrow indicates the heterozygous nucleotides of C/T. (B) A schematic diagram of GATA4 shows the location of the p.R311W mutation. (C) Multiple sequence alignment of the GATA4 indicates that residue 311 is highly evolutionarily conserved in mammals.</p

Disease-causing potential of the GATA4 mutation.

<p>(A) Functional significance of mutation prediction done by SIFT, PolyPhen-2 and MutationTaster. (B) Three-dimensional structure of GATA4 protein predicted by Swiss-PdbViewer. Upper panel is the reference structure; the lower panel is with arginine in position 311 mutated to tryptophan. (C) Double GATA4 bound to a tandem GATA4 binding site. Amino acid in position 311 is highlighted as yellow cube. (D) A schematic diagram of GATA4 protein.</p

Expression levels of GATA4 wide-type and p.R311W mutant protein in Hela cells.

<p>Western blot exhibits equal amount of GATA4 p.R311W protein as compared to the wild-type. Protein loading is normalized by the level of GAPDH.</p

Normal subcellular distribution of GATA4 mutant protein.

<p>The immunofluorescence staining of the transfected HeLa cells shows that the GATA4 p.R311W mutant protein doesn’t change its nuclear distribution.</p

Decreased transcriptional activity of GATA4 mutant protein.

<p>Relative luciferase activation of ANF-Luc in Hela cells shows significantly reduced transcriptional activity of the p.R311W mutant protein. (*, P<0.05).</p

Additional file 2: of Accurate diagnosis of spinal muscular atrophy and 22q11.2 deletion syndrome using limited deoxynucleotide triphosphates and high-resolution melting

22q11.2 region copy number determination by restricted dNTPs/HRM and MLPA. Figure S4. CLTCL1 copy number determination by restricted dNTPs and multiplex PCR. Figure S5. PI4KA/KLHL22 copy number determination by restricted dNTPs and multiplex PCR. Figure S6. MLPA results for normal control and 22q11.2 deletion samples. Table S2. Comparison of 22q11.2 detection results of Limited dNTPs/HRM and MLPA. (PDF 1098 kb

Additional file 1: of Accurate diagnosis of spinal muscular atrophy and 22q11.2 deletion syndrome using limited deoxynucleotide triphosphates and high-resolution melting

Restricted dNTPs /HRM and MLPA results for SMN1 exon7 copy number assessment. Figure S1-S2. SMN1 copy number determination by restricted dNTPs and multiplex PCR. Figure S3. MLPA results for normal control, SMA carrier and SMA samples. Table S1. Comparison of SMN1 detection results of Limited dNTPs/HRM and MLPA. (PDF 1052 kb

Data_Sheet_1_Molecular Characteristics of Community-Associated Staphylococcus aureus Isolates From Pediatric Patients With Bloodstream Infections Between 2012 and 2017 in Shanghai, China.xls

<p>Staphylococcus aureus is known as an invasive human pathogen, resulting in significant morbidity and mortality worldwide; however, information on community-associated S. aureus (CA-SA) from bloodstream infections (BSI) in children in China remains scarce. This study aimed to investigate the molecular characteristics of 78 CA-SA isolates recovered from pediatric patients with BSI between 2012 and 2017 in Shanghai. All isolates including 51 (65.4%) methicillin-susceptible S. aureus (MSSA) and 27 (34.6%) methicillin-resistant S. aureus (MRSA) isolates were characterized based on antimicrobial resistance, virulence genes, multilocus sequence typing (MLST), spa, and SCCmec typing. A total of 18 distinct sequence types (STs) and 44 spa types were identified. ST188 and ST7 were the predominant MSSA clones and ST59-MRSA-SCCmecIV/V was the most common MRSA clone. Spa t189 (9.0%, 7/78) was the most common spa type. SCCmec types IV and V were observed at frequencies of 59.3 and 40.7%, respectively. Notably, 40 (51.3%) S. aureus BSI strains were multidrug resistant (MDR), and these were mostly resistant to penicillin, erythromycin, and clindamycin. MRSA strains were associated with substantially higher rates of resistance to multiple antibiotics than MSSA strains. Fifty (64.1%, 50/78) isolates, including 19 (70.3%) MRSA isolates, harbored ≥ 10 tested virulence genes, as evaluated in this study. Ten (37.0%) MRSA isolates and four (7.8%) MSSA isolates harbored the gene encoding Panton–Valentine leukocidin (PVL). Virulence genes analysis showed diversity in different clones; the seb-sek-seq genes were present in all ST59 strains, whereas the seg-sei-sem-sen-seo genes were present in different clones including ST5, ST20, ST22, ST25, ST26, ST30, ST121, and ST487 strains. In conclusion, this study revealed that community-associated S. aureus strains from BSI in children demonstrated considerable genetic diversity, and identified major genotypes of CA-MRSA and CA-MSSA, with a high prevalence of CA-MRSA. Furthermore, major genotypes were frequently associated with specific antimicrobial resistance and toxin gene profiles. Understanding the molecular characteristics of those strains might provide further insights regarding the spread of BSI S. aureus among children between communities in China.</p

Data_Sheet_2_Molecular Characteristics of Community-Associated Staphylococcus aureus Isolates From Pediatric Patients With Bloodstream Infections Between 2012 and 2017 in Shanghai, China.xls

<p>Staphylococcus aureus is known as an invasive human pathogen, resulting in significant morbidity and mortality worldwide; however, information on community-associated S. aureus (CA-SA) from bloodstream infections (BSI) in children in China remains scarce. This study aimed to investigate the molecular characteristics of 78 CA-SA isolates recovered from pediatric patients with BSI between 2012 and 2017 in Shanghai. All isolates including 51 (65.4%) methicillin-susceptible S. aureus (MSSA) and 27 (34.6%) methicillin-resistant S. aureus (MRSA) isolates were characterized based on antimicrobial resistance, virulence genes, multilocus sequence typing (MLST), spa, and SCCmec typing. A total of 18 distinct sequence types (STs) and 44 spa types were identified. ST188 and ST7 were the predominant MSSA clones and ST59-MRSA-SCCmecIV/V was the most common MRSA clone. Spa t189 (9.0%, 7/78) was the most common spa type. SCCmec types IV and V were observed at frequencies of 59.3 and 40.7%, respectively. Notably, 40 (51.3%) S. aureus BSI strains were multidrug resistant (MDR), and these were mostly resistant to penicillin, erythromycin, and clindamycin. MRSA strains were associated with substantially higher rates of resistance to multiple antibiotics than MSSA strains. Fifty (64.1%, 50/78) isolates, including 19 (70.3%) MRSA isolates, harbored ≥ 10 tested virulence genes, as evaluated in this study. Ten (37.0%) MRSA isolates and four (7.8%) MSSA isolates harbored the gene encoding Panton–Valentine leukocidin (PVL). Virulence genes analysis showed diversity in different clones; the seb-sek-seq genes were present in all ST59 strains, whereas the seg-sei-sem-sen-seo genes were present in different clones including ST5, ST20, ST22, ST25, ST26, ST30, ST121, and ST487 strains. In conclusion, this study revealed that community-associated S. aureus strains from BSI in children demonstrated considerable genetic diversity, and identified major genotypes of CA-MRSA and CA-MSSA, with a high prevalence of CA-MRSA. Furthermore, major genotypes were frequently associated with specific antimicrobial resistance and toxin gene profiles. Understanding the molecular characteristics of those strains might provide further insights regarding the spread of BSI S. aureus among children between communities in China.</p