Ri-Tai Huang

A novel NKX2.5 loss-of-function mutation responsible for familial atrial fibrillation

Atrial fibrillation (AF) is the most commonly sustained cardiac arrhythmia, and confers a substantially increased risk of morbidity and mortality. Increasing evidence has indicated that hereditary defects are implicated in AF. However, AF is genetically heterogeneous and the genetic etiology of AF in a significant portion of patients remains unclear. In this study, the entire coding sequence and splice junctions of the GATA6 gene, which encodes a zinc-finger transcription factor crucial for cardiogenesis, were sequenced in 140 unrelated patients with lone AF. The available relatives of the index patient carrying an identified mutation and 200 unrelated ethnically-matched healthy individuals used as the controls were genotyped. The functional characteristics of the mutant GATA6 were assessed in contrast to its wild-type counterpart using a luciferase reporter assay system. As a result, a novel heterozygous GATA6 mutation, p.G469V, was identified in a family with AF inherited in an autosomal dominant pattern. The mutation was absent in the 200 control individuals and the altered amino acid was completely conserved across species. Functional analysis demonstrated that the GATA6 mutation was associated with a significantly decreased transcriptional activity. The findings provide novel insight into the molecular mechanism involved in the pathogenesis of AF, as well as insight into potential therapies for the prevention and treatment of AF.

GATA4 loss-of-function mutations underlie familial tetralogy of fallot.

Tetralogy of Fallot (TOF) represents the most common form of cyanotic congenital heart disease and accounts for significant morbidity and mortality in humans. Emerging evidence has implicated genetic defects in the pathogenesis of TOF. However, TOF is genetically heterogeneous and the genetic basis for TOF in most patients remains unclear. In this study, the GATA4 gene were sequenced in 52 probands with familial TOF, and three novel heterozygous mutations, including A9P and L51V both located in the putative first transactivational domain and N285S in the C‐terminal zinc finger, were identified in three probands, respectively. Genetic analysis of the pedigrees demonstrated that in each family the mutation cosegregated with TOF with complete penetrance. The missense mutations were absent in 800 control chromosomes and the altered amino acids were highly conserved evolutionarily. Functional analysis showed that the GATA4 mutants were consistently associated with diminished DNA‐binding affinity and decreased transcriptional activity. Furthermore, the N285S mutation completely disrupted the physical interaction between GATA4 and TBX5. To our knowledge, this report associates GATA4 loss‐of‐function mutations with familial TOF for the first time, providing novel insight into the molecular mechanism involved in TOF and suggesting potential implications for the early prophylaxis and allele‐specific therapy of TOF.

A novel HAND2 loss-of-function mutation responsible for tetralogy of Fallot

Congenital heart disease (CHD), the most common type of developmental abnormality, is associated with substantial morbidity and mortality in humans worldwide. The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed 2 (HAND2), has been demonstrated to be crucial for normal cardiovascular development in animal models. However, whether a genetically defective HAND2 contributes to congenital heart disease (CHD) in humans remains to be explored. In this study, the entire coding region and splicing boundaries of the HAND2 gene were sequenced in a cohort of 145 unrelated patients with CHD. A total of 200 unrelated, ethnically-matched healthy individuals used as controls were also genotyped for HAND2. The functional effect of the mutant HAND2 was characterized in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. As a result, a novel heterozygous HAND2 mutation, p.L47P, was identified in a patient with tetralogy of Fallot (TOF). The misense mutation, which altered the amino acid conserved evolutionarily among species, was absent in 400 control chromosomes. Functional analyses unveiled that the mutant HAND2 had a significantly decreased transcriptional activity. Furthermore, the mutation markedly reduced the synergistic activation between HAND2 and GATA4 or NKX2.5, other two cardiac key transcription factors involved in the pathogenesis of CHD. To the best of our knowledge, this study is the first to report the association of a HAND2 loss-of-function mutation with an increased vulnerability to TOF in humans, which provides novel insight into the molecular mechanism underpinning CHD, suggesting potential implications for the genetic counseling of families with CHD.

PITX2 loss-of-function mutation contributes to tetralogy of Fallot.

Congenital heart disease (CHD) is the most prevalent developmental abnormality in humans and is the most common non-infectious cause of infant morbidity and mortality. Increasing evidence demonstrates that genetic defects are involved in the pathogenesis of CHD. However, CHD is genetically heterogeneous, and the genetic determinants underpinning CHD in most patients remain unknown. In this study, the whole coding region of the PITX2 gene (isoform c) was sequenced in 185 unrelated patients with CHD. The available relatives of a mutation carrier and 300 unrelated healthy individuals used as controls were also genotyped for PITX2. The functional characteristics of the mutation were delineated by using a dual-luciferase reporter assay system. As a result, a novel heterozygous PITX2 mutation, p.Q102L, was identified in a patient with tetralogy of Fallot (TOF). Genetic analysis of the index patients pedigree showed that the mutation co-segregated with TOF. The mutation was absent in 600 reference chromosomes. Biochemical analysis revealed that the Q102L-mutant PITX2 is associated with significantly reduced transcriptional activity compared with its wild-type counterpart. Furthermore, the mutation markedly decreased the synergistic activation between PITX2 and NKX2-5. This study firstly associates PITX2 loss-of-function mutation with increased susceptibility to TOF, providing novel insight into the molecular mechanism of CHD.

Somatic GATA5 mutations in sporadic tetralogy of Fallot

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease, with high morbidity and mortality rates. Accumulating evidence has demonstrated that genetic defects play an important role in the pathogenesis of TOF. However, the molecular basis of TOF in the majority of patients remains to be determined. In the present study, sequence analysis of the coding exons and exon-intron boundaries of GATA5, a gene encoding a zinc finger‑containing transcriptional factor crucial for cardiogenesis, was performed on genomic DNA isolated from resected cardiac tissue and matched blood samples of 85 unrelated patients who underwent surgical repair of TOF. Genotyping was performed on the cardiac tissue and matched blood samples from 63 unrelated patients who underwent cardiac valve replacement due to rheumatic heart disease as well as the blood samples obtained from 200 unrelated healthy individuals. The functional effect of the mutations was evaluated by using a luciferase reporter assay system. As a result, the novel heterozygous GATA5 mutations, p.D203E and p.Y208X, were found in the cardiac tissues of two TOF patients, respectively. There were no mutations in the cardiac tissues obtained from 63 patients with rheumatic heart disease nor in the blood samples obtained from the 348 subjects. Functional analysis revealed that the GATA5 mutants were consistently associated with significantly decreased transcriptional activity compared with their wild-type counterpart. Thus, results of this study showed an association of somatic GATA5 mutations with TOF, providing further insight into the underlying molecular mechanism of TOF.

A HAND2 Loss-of-Function Mutation Causes Familial Ventricular Septal Defect and Pulmonary Stenosis

Congenital heart disease (CHD) is the most common developmental abnormality, and is the leading noninfectious cause of mortality in neonates. Increasing evidence demonstrates that genetic defects play an important role in the pathogenesis of CHD. However, CHD exhibits substantial heterogeneity, and the genetic determinants for CHD remain unknown in the overwhelming majority of cases. In the current study, the coding exons and flanking introns of the HAND2 gene, which encodes a basic helix-loop-helix transcription factor essential for normal cardiovascular development, were sequenced in 192 unrelated patients with CHD, and a novel heterozygous mutation, p.S65I, was identified in a patient with congenital ventricular septal defect (VSD). Genetic analysis of the index patient’s pedigree revealed that the mutation was present in all seven affected family members available, but absent in the 13 unaffected family members examined. Besides, in addition to VSD, five of the proband’s close relatives also had pulmonary stenosis (PS), and the proband’s son also had double outlet right ventricle (DORV). The missense mutation, which altered an evolutionarily conserved amino acid, was absent in 300 unrelated, ethnically matched healthy individuals. Biological analyses using a dual-luciferase reporter assay system showed that the mutant HAND2 was associated with significantly diminished transcriptional activity. Furthermore, the mutation abolished the synergistic activation between HAND2 and GATA4, as well as NKX2.5—two other cardiac core transcriptional factors that have been causally linked to CHD. These findings indicate that HAND2 loss-of-function mutation contributes to human CHD, perhaps via its interaction with GATA4 and NKX2.5.

A Novel NKX2.6 Mutation Associated with Congenital Ventricular Septal Defect

Congenital heart disease (CHD) is the most common birth defect and is the most prevalent non-infectious cause of infant death. Aggregating evidence demonstrates that genetic defects are involved in the pathogenesis of CHD. However, CHD is genetically heterogeneous and the genetic determinants for CHD in an overwhelming majority of patients remain unknown. In this study, the coding regions and splice junctions of the NKX2.6 gene, which encodes a homeodomain transcription factor crucial for cardiovascular development, were sequenced in 210 unrelated CHD patients. As a result, a novel heterozygous NKX2.6 mutation, p.K152Q, was identified in an index patient with ventricular septal defect (VSD). Genetic analysis of the proband’s available family members showed that the mutation cosegregated with VSD transmitted as an autosomal dominant trait with complete penetrance. The missense mutation was absent in 400 control chromosomes and the altered amino acid was completely conserved evolutionarily across species. Due to unknown transcriptional targets of NKX2.6, the functional characteristics of the identified mutation at transcriptional activity were analyzed by using NKX2.5 as a surrogate. Alignment between human NKX2.6 and NKX2.5 proteins displayed that K152Q-mutant NKX2.6 was equivalent to K158Q-mutant NKX2.5, and introduction of K158Q into NKX2.5 significantly reduced its transcriptional activating function when compared with its wild-type counterpart. This study firstly links NKX2.6 loss-of-function mutation with increased susceptibility to isolated VSD, providing novel insight into the molecular mechanism underpinning VSD and contributing to the development of new preventive and therapeutic strategies for this common form of CHD.

TBX20 loss-of-function mutation responsible for familial tetralogy of Fallot or sporadic persistent truncus arteriosus

Congenital heart disease (CHD), the most common form of developmental abnormality in humans, remains a leading cause of morbidity and mortality in neonates. Genetic defects have been recognized as the predominant causes of CHD. Nevertheless, CHD is of substantial genetic heterogeneity and the genetic defects underlying CHD in most cases remain unclear. In the current study, the coding regions and splicing junction sites of the TBX20 gene, which encodes a T-box transcription factor key to cardiovascular morphogenesis, were sequenced in 175 unrelated patients with CHD, and a novel heterozygous TBX20 mutation, p.K274X, was identified in an index patient with tetralogy of Fallot (TOF). Genetic analysis of the probands available family members showed that his father, elder brother and son had also TOF. In addition, his father and elder brother had also atrial septal defect, and his niece had persistent truncus arteriosus and ventricular septal defect. Analysis of the pedigree revealed that the mutation co-segregated with CHD transmitted in an autosomal dominant fashion, with complete penetrance. The nonsense mutation, which was absent in the 800 control chromosomes, was predicted to produce a truncated protein with only the amino terminus and partial T-box domain left. Functional analyses by using a dual-luciferase reporter assay system showed that the mutant TBX20 lost the ability to transactivate the target gene ANF. Furthermore, the mutation reduced the synergistic activation between TBX20 and NKX2.5 as well as GATA4, two other transcriptional factors previously associated with various CHD, encompassing TOF. This study firstly links TBX20 loss-of-function mutation to familial TOF or sporadic persistent truncus arteriosus, providing novel insight into the molecular pathogenesis of CHD.

CASZ1 loss-of-function mutation associated with congenital heart disease

As the most common form of birth defect in humans, congenital heart disease (CHD) is associated with substantial morbidity and mortality in both children and adults. Increasing evidence demonstrates that genetic defects play a pivotal role in the pathogenesis of CHD. However, CHD is of great heterogeneity, and in an overwhelming majority of cases, the genetic determinants underpinning CHD remain elusive. In the present investigation, the coding exons and flanking introns of the CASZ1 gene, which codes for a zinc finger transcription factor essential for the cardiovascular morphogenesis, were sequenced in 172 unrelated patients with CHD. As a result, a novel heterozygous CASZ1 mutation, p.L38P, was identified in an index patient with congenital ventricular septal defect (VSD). Genetic scanning of the mutation carriers available family members revealed that the mutation was present in all affected patients but absent in unaffected individuals. Analysis of the probands pedigree showed that the mutation co-segregated with VSD, which was transmitted as an autosomal dominant trait with complete penetrance. The missense mutation, which altered the amino acid that was highly conserved evolutionarily, was absent in 200 unrelated, ethnically-matched healthy subjects used as controls. Functional deciphers by using a dual-luciferase reporter assay system unveiled that the mutant CASZ1 had significantly reduced transcriptional activity as compared with its wild-type counterpart. To the best of our knowledge, the current study firstly identifies CASZ1 as a new gene predisposing to CHD in humans, which provides novel insight into the molecular mechanisms underlying CHD and a potential therapeutic target for CASZ1-associated CHD, suggesting potential implications for personalized prophylaxis and therapy of CHD.

CASZ1 loss-of-function mutation contributes to familial dilated cardiomyopathy.

Abstract Background: The zinc finger transcription factor CASZ1 plays a key role in cardiac development and postnatal adaptation, and in mice, deletion of the CASZ1 gene leads to dilated cardiomyopathy (DCM). However, in humans whether genetically defective CASZ1 contributes to DCM remains unclear. Methods: The coding exons and splicing junction sites of the CASZ1 gene were sequenced in 138 unrelated patients with idiopathic DCM. The available family members of the index patient harboring an identified CASZ1 mutation and 200 unrelated, ethnically matched healthy individuals used as controls were genotyped for CASZ1. The functional characteristics of the mutant CASZ1 were analyzed in contrast to its wild-type counterpart using a luciferase reporter assay system. Results: A novel heterozygous CASZ1 mutation, p.K351X, was identified in an index patient with DCM. Genetic analysis of the mutation carrier’s family showed that the mutation co-segregated with DCM, which was transmitted in an autosomal dominant pattern with complete penetrance. The nonsense mutation, which was absent in 400 referential chromosomes, altered the amino acid that was highly conserved evolutionarily. Biological investigations revealed that the mutant CASZ1 had no transcriptional activity. Conclusions: The current study reveals CASZ1 as a new gene responsible for human DCM, which provides novel mechanistic insight and potential therapeutic target for CASZ1-associated DCM, implying potential implications in improved prophylactic and therapeutic strategies for DCM, the most common type of primary myocardial disease.