应用PCR技术对先天性长QT综合征KCNQ1 基因进行定点突变的研究

利用聚合酶链反应（PCR）技术对长QT综合征（LQTS）KCNQ1基因进行定点突变的研究。首先设计两对引物（包含预定的突变）,通过3轮PCR扩增,扩增出含有所需突变位点的片段,然后将片段克隆入T载体中,通过酶切连接的方法将突变点引入到pIRES2-EGFP-KCNQ1中,随后用Effectene转染试剂介导转染HEK293细胞。结果在真核表达载体pIRES2-EGFP-KCNQ1基础上获得了KCNQ1 cDNA C934T的突变体,测序表明在序列中发生了预期的突变。将含突变点的pIRES2-EGFP-KCNQ1转染HEK293细胞后,在荧光显微镜下观察到被转染的HEK293细胞发出绿色荧光,表明含突变点的pIRES2-EGFP-KCNQ1得到了表达。Abstract: To study PCR site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro. The site-directed mutagenesis of LQTS gene KCNQ1 was made by PCR. Two sets of primers were designed according to the sequence of KCNQ1 cDNA, and mismatch was introduced into primers. Mutagenesis was performed in a three-step PCR. The amplified fragments from the third PCR which contained the mutation site were subcloned into the T-vecor PCR2.1.Then the fragments containing the mutation site was obtained from PCR2.1 with restriction enzyme digestion and was inserted into the same restriction site of pIRES2-EGFP-KCNQ1. With Effectene Transfection Reagent, pIRES2-EGFP-KCNQ1 was transfected into HEK293 cell. The sequencing analysis showed that the mutation site was correct. Mutation from T to C in 934 site of KCNQ1 cDNA was found. Under the fluorescence microscope, the green fluorescence was spread in the transfected HEK293 cell, meaning the pIRES2-EGFP-KCNQ1 containing the mutation site was expressed correctly.     

Mechanistic Basis for Type 2 Long QT Syndrome Caused by KCNH2 Mutations that Disrupt Conserved Arginine Residues in the Voltage Sensor

KCNH2 encodes the Kv11.1 channel, which conducts the rapidly activating delayed rectifier K⁺ current (I _Kr) in the heart. KCNH2 mutations cause type 2 long QT syndrome (LQT2), which increases the risk for life-threatening ventricular arrhythmias. LQT2 mutations are predicted to prolong the cardiac action potential (AP) by reducing I _Kr during repolarization. Kv11.1 contains several conserved basic amino acids in the fourth transmembrane segment (S4) of the voltage sensor that are important for normal channel trafficking and gating. This study sought to determine the mechanism(s) by which LQT2 mutations at conserved arginine residues in S4 (R531Q, R531W or R534L) alter Kv11.1 function. Western blot analyses of HEK293 cells transiently expressing R531Q, R531W or R534L suggested that only R534L inhibited Kv11.1 trafficking. Voltage-clamping experiments showed that R531Q or R531W dramatically altered Kv11.1 current (I _Kv11.1) activation, inactivation, recovery from inactivation and deactivation. Coexpression of wild type (to mimic the patients’ genotypes) mostly corrected the changes in I _Kv11.1 activation and inactivation, but deactivation kinetics were still faster. Computational simulations using a human ventricular AP model showed that accelerating deactivation rates was sufficient to prolong the AP, but these effects were minimal compared to simply reducing I _Kr. These are the first data to demonstrate that coexpressing wild type can correct activation and inactivation dysfunction caused by mutations at a critical voltage-sensing residue in Kv11.1. We conclude that some Kv11.1 mutations might accelerate deactivation to cause LQT2 but that the ventricular AP duration is much more sensitive to mutations that decrease I _Kr. This likely explains why most LQT2 mutations are nonsense or trafficking-deficient.     

Mechanisms of Premature Ventricular Complexes Caused by QT Prolongation

QT prolongation, due to lengthening of the action potential duration in the ventricles, is a major risk factor of lethal ventricular arrhythmias. A widely known consequence of QT prolongation is the genesis of early afterdepolarizations (EADs), which are associated with arrhythmias through the generation of premature ventricular complexes (PVCs). However, the vast majority of the EADs observed experimentally in isolated ventricular myocytes are phase-2 EADs, and whether phase-2 EADs are mechanistically linked to PVCs in cardiac tissue remains an unanswered question. In this study, we investigate the genesis of PVCs using computer simulations with eight different ventricular action potential models of various species. Based on our results, we classify PVCs as arising from two distinct mechanisms: repolarization gradient (RG)-induced PVCs and phase-2 EAD-induced PVCs. The RG-induced PVCs are promoted by increasing RG and L-type calcium current and are insensitive to gap junction coupling. EADs are not required for this PVC mechanism. In a paced beat, a single or multiple PVCs can occur depending on the properties of the RG. In contrast, phase-2 EAD-induced PVCs occur only when the RG is small and are suppressed by increasing RG and more sensitive to gap junction coupling. Unlike with RG-induced PVCs, in each paced beat, only a single EAD-induced PVC can occur no matter how many EADs in an action potential. In the wide parameter ranges we explore, RG-induced PVCs can be observed in all models, but the EAD-induced PVCs can only be observed in five of the eight models. The links between these two distinct PVC mechanisms and arrhythmogenesis in animal experiments and clinical settings are discussed.     

Autosomal-Dominant Multiple Pterygium Syndrome Is Caused by Mutations in MYH3

Multiple pterygium syndrome (MPS) is a phenotypically and genetically heterogeneous group of rare Mendelian conditions characterized by multiple pterygia, scoliosis, and congenital contractures of the limbs. MPS typically segregates as an autosomal-recessive disorder, but rare instances of autosomal-dominant transmission have been reported. Whereas several mutations causing recessive MPS have been identified, the genetic basis of dominant MPS remains unknown. We identified four families affected by dominantly transmitted MPS characterized by pterygia, camptodactyly of the hands, vertebral fusions, and scoliosis. Exome sequencing identified predicted protein-altering mutations in embryonic myosin heavy chain (MYH3) in three families. MYH3 mutations underlie distal arthrogryposis types 1, 2A, and 2B, but all mutations reported to date occur in the head and neck domains. In contrast, two of the mutations found to cause MPS in this study occurred in the tail domain. The phenotypic overlap among persons with MPS, coupled with physical findings distinct from other conditions caused by mutations in MYH3, suggests that the developmental mechanism underlying MPS differs from that of other conditions and/or that certain functions of embryonic myosin might be perturbed by disruption of specific residues and/or domains. Moreover, the vertebral fusions in persons with MPS, coupled with evidence of MYH3 expression in bone, suggest that embryonic myosin plays a role in skeletal development.     

Mutation analysis in congenital Long QT Syndrome--a case with missense mutations in KCNQ1 and SCN5A

Long QT Syndrome (LQTS) is a cardiac disease characterized by a prolonged QT interval on a surface electrocardiogram (ECG) and by clinical symptoms such as seizures, syncope, and cardiac sudden death. At present, causal mutations of LQTS have been identified in five cardiac ion-channel genes. Because a causal mutation is usually unique to a specific family and can be located in any region of any of these five genes, a mutation analysis effort may require screening of the complete coding regions of each of these genes. The causative nature of a detected mutation can then be determined either by family history or by functional studies, such as the electrophysiological signature of the mutation. Here we describe a mutation analysis of an LQTS patient who carries two heterozygous missense mutations in two different LQTS genes. The first mutation identified, A572D in SCN5A, was not linked with clinical LQTS features in the two other mutation carriers in the family; neither was it identified in 90 healthy controls. Therefore, this mutation most likely has either a mild effect on cardiac ion-channel function or represents a very rare polymorphism. The second mutation, V254M in KCNQ1, co-segregated with higher QT intervals and symptoms in other family members, and was previously reported in another LQTS family. Because the clinical LQTS symptoms are most pronounced in the proband, a combined effect of both mutations cannot be excluded, although no functional data are available to support such an hypothesis. We conclude that, for newly presented LQTS cases, a mutation analysis strategy should routinely screen the complete coding region of all LQTS genes, followed by an evaluation of the identified mutation(s) in conjunction with family or functional data.     

Altered KCNQ3 Potassium Channel Function Caused by the W309R Pore-Helix Mutation Found in Human Epilepsy

The second tryptophan (W) residue of the conserved WW motif in the pore helix of many K⁺ channel subunit is thought to interact with the tyrosine (Y) residues of the selectivity filter. A missense mutation causing the replacement of the corresponding residues with an arginine (W309R) occurs in KCNQ3 subunits forming part of M-channels. In this study, we examined the functional consequences of the W309R mutation in heterogously expressed KCNQ channels. Homomeric KCNQ3^W309R channels lacked KCNQ currents. Heteromeric KCNQ2/KCNQ3^W309R channels displayed a dominant-negative suppression of current and a significant modification in gating properties when compared with heteromeric KCNQ3/KCNQ2 channels mimicking the M-channels. A three-dimensional homology model in the W309R mutant indicated that the R side chain of pore helices is too far from the Y side chain of the selectivity filter to interact via hydrogen bonds with each other and stabilize the pore structure. Collectively, the present results suggest that the second W residues of pore helices and their chemical interaction with the Y residues of the selectivity filter are essential for normal K⁺ channel function. This pore-helix mutation, if occurs in the brain M channels, could thus lead to a channel dysfunction sufficient to trigger epileptic hyperexcitability.     

Observational Cohort Study of Ventricular Arrhythmia in Adults with Marfan Syndrome Caused by FBN1 Mutations

Background

Marfan syndrome is associated with ventricular arrhythmia but risk factors including FBN1 mutation characteristics require elucidation.

Methods and Results

We performed an observational cohort study of 80 consecutive adults (30 men, 50 women aged 42±15 years) with Marfan syndrome caused by FBN1 mutations. We assessed ventricular arrhythmia on baseline ambulatory electrocardiography as >10 premature ventricular complexes per hour (>10 PVC/h), as ventricular couplets (Couplet), or as non-sustained ventricular tachycardia (nsVT), and during 31±18 months of follow-up as ventricular tachycardia (VT) events (VTE) such as sudden cardiac death (SCD), and sustained ventricular tachycardia (sVT). We identified >10 PVC/h in 28 (35%), Couplet/nsVT in 32 (40%), and VTE in 6 patients (8%), including 3 with SCD (4%). PVC>10/h, Couplet/nsVT, and VTE exhibited increased N-terminal pro–brain natriuretic peptide serum levels(P<.001). All arrhythmias related to increased NT-proBNP (P<.001), where PVC>10/h and Couplet/nsVT also related to increased indexed end-systolic LV diameters (P = .024 and P = .020), to moderate mitral valve regurgitation (P = .018 and P = .003), and to prolonged QTc intervals (P = .001 and P = .006), respectively. Moreover, VTE related to mutations in exons 24–32 (P = .021). Kaplan–Meier analysis corroborated an association of VTE with increased NT-proBNP (P<.001) and with mutations in exons 24–32 (P<.001).

Conclusions

Marfan syndrome with causative FBN1 mutations is associated with an increased risk for arrhythmia, and affected persons may require life-long monitoring. Ventricular arrhythmia on electrocardiography, signs of myocardial dysfunction and mutations in exons 24–32 may be risk factors of VTE.     

QT延长综合征的分子生物学机制

遗传性QT延长综合征又称突发性LQTS,是一种以QT间期延长(LQT)、突发性昏厥、惊厥、甚至猝死为特征的先天性心脏病。该将阐述其机制及其研究进展。     

Heimler Syndrome Is Caused by Hypomorphic Mutations in the Peroxisome-Biogenesis Genes PEX1 and PEX6

Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities, and occasional or late-onset retinal pigmentation. We ascertained eight families affected by HS and, by using a whole-exome sequencing approach, identified biallelic mutations in PEX1 or PEX6 in six of them. Loss-of-function mutations in both genes are known causes of a spectrum of autosomal-recessive peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome. PBDs are characterized by leukodystrophy, hypotonia, SNHL, retinopathy, and skeletal, craniofacial, and liver abnormalities. We demonstrate that each HS-affected family has at least one hypomorphic allele that results in extremely mild peroxisomal dysfunction. Although individuals with HS share some subtle clinical features found in PBDs, the diagnosis was not suggested by routine blood and skin fibroblast analyses used to detect PBDs. In conclusion, our findings define HS as a mild PBD, expanding the pleiotropy of mutations in PEX1 and PEX6.     

Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

Congenital long QT syndrome (LQTS) is a hereditary disorder that leads to sudden cardiac death secondary to fatal cardiac arrhythmias. Although many genes for LQTS have been described, the etiology remains unknown in 30%–40% of cases. In the present study, a large Chinese family (four generations, 49 individuals) with autosomal-dominant LQTS was clinically evaluated. Genome-wide linkage analysis was performed by using polymorphic microsatellite markers to map the genetic locus, and positional candidate genes were screened by sequencing for mutations. The expression pattern and functional characteristics of the mutated protein were investigated by western blotting and patch-clamp electrophysiology. The genetic locus of the LQTS-associated gene was mapped to chromosome 11q23.3-24.3. A heterozygous mutation (Kir3.4-Gly387Arg) was identified in the G protein-coupled, inwardly rectifying potassium channel subunit Kir3.4, encoded by the KCNJ5 gene. The Kir3.4-Gly387Arg mutation was present in all nine affected family members and absent in 528 ethnically matched controls. Western blotting of human cardiac tissue demonstrated significant Kir3.4 expression levels in the cardiac ventricles. Heterologous expression studies with Kir3.4-Gly387Arg revealed a loss-of-function electrophysiological phenotype resulting from reduced plasma membrane expression. Our findings suggest a role for Kir3.4 in the etiology of LQTS.     

Isoform-Specific Dominant-Negative Effects Associated with hERG1 G628S Mutation in Long QT Syndrome

Background

Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K⁺ channel with properties similar to the rapidly activating delayed rectifying K⁺ current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1a_USO isoforms.

Methods

A double-stable, mammalian expression system was developed to characterize isoform-specific dominant-negative effects of G628S-containing channels when co-expressed at equivalent levels with wild-type hERG1a. Western blot and co-immunoprecipitation studies were performed to study the trafficking and co-assembly of wild-type and mutant hERG1 isoforms. Patch-clamp electrophysiology was performed to characterize hERG1 channel function and the isoform-specific dominant-negative effects associated with the G628S mutation.

Conclusions

The non-functional hERG1a-G628S and hERG1b-G628S channels co-assembled with wild-type hERG1a and dominantly suppressed hERG1 current. In contrast, G628S-induced dominant-negative effects were absent in the context of the hERG1a_USO isoform. hERG1a_USO-G628S channels did not appreciably associate with hERG1a and did not significantly suppress hERG1 current when co-expressed at equivalent ratios or at ratios that approximate those found in cardiac tissue. These results suggest that the dominant-negative effects of LQT2 mutations may primarily occur in the context of the hERG1a and hERG1b isoforms.     

Novel Mutations Found in Two Genes of Thai Patients with Isolated Methylmalonic Acidemia

Molecular genetic analysis of three patients diagnosed with isolated methylmalonic acidemia (MMA) revealed that one was mut ⁰ MMA, with a mutation in the MUT gene encoding the l-methylmalonyl-CoA mutase (MCM), and two were cblB MMA, with mutations in the MMAB gene required for synthesizing the deoxyadenosylcobalamin cofactor of MCM. The mut ⁰ patient was homozygous for a novel nonsense mutation in MUT, p.R31X (c.167C → T), and heterozygous for three previously described polymorphisms, p.K212K (c.712A → G), p.H532R (c.1671A → G), and p.V671I (c.2087G → A). The new MMAB mutation, p.E152X (c.454G → T), was found to be homozygous in one cblB patient and heterozygous in the other patient, who also had four intron polymorphisms in this gene.     

Neu-Laxova Syndrome,an Inborn Error of Serine Metabolism,Is Caused by Mutations in PHGDH

Neu-Laxova syndrome (NLS) is a rare autosomal-recessive disorder characterized by severe fetal growth restriction, microcephaly, a distinct facial appearance, ichthyosis, skeletal anomalies, and perinatal lethality. The pathogenesis of NLS remains unclear despite extensive clinical and pathological phenotyping of the >70 affected individuals reported to date, emphasizing the need to identify the underlying genetic etiology, which remains unknown. In order to identify the cause of NLS, we conducted a positional-mapping study combining autozygosity mapping and whole-exome sequencing in three consanguineous families affected by NLS. Surprisingly, the NLS-associated locus identified in this study was solved at the gene level to reveal mutations in PHGDH, which is known to be mutated in individuals with microcephaly and developmental delay. PHGDH encodes the first enzyme in the phosphorylated pathway of de novo serine synthesis, and complete deficiency of its mouse ortholog recapitulates many of the key features of NLS. This study shows that NLS represents the extreme end of a known inborn error of serine metabolism and highlights the power of genomic sequencing in revealing the unsuspected allelic nature of apparently distinct clinical entities.     

Keppen-Lubinsky Syndrome Is Caused by Mutations in the Inwardly Rectifying K+ Channel Encoded by KCNJ6

Keppen-Lubinsky syndrome (KPLBS) is a rare disease mainly characterized by severe developmental delay and intellectual disability, microcephaly, large prominent eyes, a narrow nasal bridge, a tented upper lip, a high palate, an open mouth, tightly adherent skin, an aged appearance, and severe generalized lipodystrophy. We sequenced the exomes of three unrelated individuals affected by KPLBS and found de novo heterozygous mutations in KCNJ6 (GIRK2), which encodes an inwardly rectifying potassium channel and maps to the Down syndrome critical region between DIRK1A and DSCR4. In particular, two individuals shared an in-frame heterozygous deletion of three nucleotides (c.455_457del) leading to the loss of one amino acid (p.Thr152del). The third individual was heterozygous for a missense mutation (c.460G>A) which introduces an amino acid change from glycine to serine (p.Gly154Ser). In agreement with animal models, the present data suggest that these mutations severely impair the correct functioning of this potassium channel. Overall, these results establish KPLBS as a channelopathy and suggest that KCNJ6 (GIRK2) could also be a candidate gene for other lipodystrophies. We hope that these results will prompt investigations in this unexplored class of inwardly rectifying K⁺ channels.     

Mechanisms underlying modulation of neuronal KCNQ2/KCNQ3 potassium channels by extracellular protons

Changes in extracellular pH occur during both physiological neuronal activity and pathological conditions such as epilepsy and stroke. Such pH changes are known to exert profound effects on neuronal activity and survival. Heteromeric KCNQ2/3 potassium channels constitute a potential target for modulation by H+ ions as they are expressed widely within the CNS and have been proposed to underlie the M-current, an important determinant of excitability in neuronal cells. Whole-cell and single-channel recordings demonstrated a modulation of heterologously expressed KCNQ2/3 channels by extracellular H+ ions. KCNQ2/3 current was inhibited by H+ ions with an IC50 of 52 nM (pH 7.3) at -60 mV, rising to 2 microM (pH 5.7) at -10 mV. Neuronal M-current exhibited a similar sensitivity. Extracellular H+ ions affected two distinct properties of KCNQ2/3 current: the maximum current attainable upon depolarization (Imax) and the voltage dependence of steady-state activation. Reduction of Imax was antagonized by extracellular K+ ions and affected by mutations within the outer-pore turret, indicating an outer-pore based process. This reduction of Imax was shown to be due primarily to a decrease in the maximum open-probability of single KCNQ2/3 channels. Single-channel open times were shortened by acidosis (pH 5.9), while closed times were increased. Acidosis also recruited a longer-lasting closed state, and caused a switch of single-channel activity from the full-conductance state ( approximately 8 pS) to a subconductance state ( approximately 5 pS). A depolarizing shift in the activation curve of macroscopic KCNQ2/3 currents and single KCNQ2/3 channels was caused by acidosis, while alkalosis caused a hyperpolarizing shift. Activation and deactivation kinetics were slowed by acidosis, indicating specific effects of H+ ions on elements involved in gating. Contrasting modulation of homomeric KCNQ2 and KCNQ3 currents revealed that high sensitivity to H+ ions was conferred by the KCNQ3 subunit.     

Mutations in NOTCH1 Cause Adams-Oliver Syndrome

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5′ region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743−1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.