Novel mutations in the TBX5 gene in patients with Holt-Oram Syndrome

The Holt-Oram syndrome (HOS) is an autosomal dominant condition characterized by upper limb and cardiac malformations. Mutations in the TBX5 gene cause HOS and have also been associated with isolated heart and arm defects. Interactions between the TBX5, GATA4 and NKX2.5 proteins have been reported in humans. We screened the TBX5, GATA4, and NKX2.5 genes for mutations, by direct sequencing, in 32 unrelated patients presenting classical (8) or atypical HOS (1), isolated congenital heart defects (16) or isolated upper-limb malformations (7). Pathogenic mutations in the TBX5 gene were found in four HOS patients, including two new mutations (c.374delG; c.678G > T) in typical patients, and the hotspot mutation c.835C > T in two patients, one of them with an atypical HOS phenotype involving lower-limb malformations. Two new mutations in the GATA4 gene were found in association with isolated upper-limb malformations, but their clinical significance remains to be established. A previously described possibly pathogenic mutation in the NKX2.5 gene (c.73C > 7) was detected in a patient with isolated heart malformations and also in his clinically normal father.     

Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in clinical practice. We first reported an S140G mutation of KCNQ1, an alpha subunit of potassium channels, in one Chinese kindred with AF. However, the molecular defects and cellular mechanisms in most patients with AF remain to be identified. We evaluated 28 unrelated Chinese kindreds with AF and sequenced eight genes of potassium channels (KCNQ1, HERG, KCNE1, KCNE2, KCNE3, KCNE4, KCNE5, and KCNJ2). An arginine-to-cysteine mutation at position 27 (R27C) of KCNE2, the beta subunit of the KCNQ1-KCNE2 channel responsible for a background potassium current, was found in 2 of the 28 probands. The mutation was present in all affected members in the two kindreds and was absent in 462 healthy unrelated Chinese subjects. Similar to KCNQ1 S140G, the mutation had a gain-of-function effect on the KCNQ1-KCNE2 channel; unlike long QT syndrome-associated KCNE2 mutations, it did not alter HERG-KCNE2 current. The mutation did not alter the functions of the HCN channel family either. Thus, KCNE2 R27C is a gain-of-function mutation associated with the initiation and/or maintenance of AF.     

p.Ala546 > Asp and p.Arg555 > Trp mutations of TGFBI gene and their clinical manifestations in two large Chinese families with granular corneal dystrophy type I

The aim of this study was to conduct clinical, genetic, and molecular analysis of Chinese patients with granular corneal dystrophy type I (CDGG1). Two large unrelated Chinese families with CDGG1 were clinically and genetically evaluated. Molecular genetic analysis was performed on DNA extracted from peripheral blood. Exons 4, 11, 12, and 14 of the human transforming growth factor beta-induced gene (TGFBI, formerly designated BIGH3) were amplified by PCR, scanned for mutations using the single-strand conformation polymorphism method, and the mutations identified by nucleotide sequencing. One family segregated the p.Ala546 > Asp mutation, and the other family had a p.Arg555 > Trp mutation. These missense mutations were not found in 53 unrelated, healthy individuals analyzed as controls. Clinical and genetic evaluations revealed the variable severity, symmetry, and age of onset in visual impairment in these families for different mutations. Penetrance of visual impairment in these families was 100% and 75%, respectively. This study confirms that the p.Arg555 > Trp mutation is a frequent cause of CDGG1, and that the p.Ala546 > Asp mutation is also associated with this disease.     

A Cypher/ZASP mutation associated with dilated cardiomyopathy alters the binding affinity to protein kinase C

Dilated cardiomyopathy is characterized by ventricular dilation with systolic dysfunction of cardiac muscle. Recent genetic studies have revealed that mutations in genes for cytoskeleton proteins distributed in the Z-disc and/or intercalated discs of the cardiac muscle are major predictors of cardiomyopathy. However, as mutations in these genes can account for only a part of the patient population, there should be another disease-causing gene(s) for cardiomyopathy. Cypher/ZASP appears to be an ideal candidate for the cardiomyopathy causative gene, because Cypher/ZASP encodes a Z-disc associated protein, and recent studies have demonstrated that Cypher/ZASP knock-out mice develop cardiomyopathy. In this study, we searched for sequence variations in Cypher/ZASP in 96 unrelated Japanese patients with dilated cardiomyopathy. A D626N mutation located within the third LIM domain was identified in a familial case but not found in the unrelated controls. A family study of the patient showed that all affected siblings tested had the same mutation. Clinical information of the affected family members suggested that the mutation was associated with late onset cardiomyopathy. To reveal the biochemical changes due to the mutation, we performed a yeast two-hybrid assay and a pull-down assay. It was demonstrated by both assays that the D626N mutation of Cypher/ZASP increased the affinity of the LIM domain for protein kinase C, suggesting a novel biochemical mechanism of the pathogenesis of dilated cardiomyopathy.     

Functional dominant-negative mutation of sodium channel subunit gene SCN3B associated with atrial fibrillation in a Chinese GeneID population

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the clinic, and accounts for more than 15% of strokes. Mutations in cardiac sodium channel α, β1 and β2 subunit genes (SCN5A, SCN1B, and SCN2B) have been identified in AF patients. We hypothesize that mutations in the sodium channel β3 subunit gene SCN3B are also associated with AF. To test this hypothesis, we carried out a large scale sequencing analysis of all coding exons and exon-intron boundaries of SCN3B in 477 AF patients (28.5% lone AF) from the GeneID Chinese Han population. A novel A130V mutation was identified in a 46-year-old patient with lone AF, and the mutation was absent in 500 controls. Mutation A130V dramatically decreased the cardiac sodium current density when expressed in HEK293/Na_v1.5 stable cell line, but did not have significant effect on kinetics of activation, inactivation, and channel recovery from inactivation. When co-expressed with wild type SCN3B, the A130V mutant SCN3B negated the function of wild type SCN3B, suggesting that A130V acts by a dominant negative mechanism. Western blot analysis with biotinylated plasma membrane protein extracts revealed that A130V did not affect cell surface expression of Na_v1.5 or SCN3B, suggesting that mutant A130V SCN3B may not inhibit sodium channel trafficking, instead may affect conduction of sodium ions due to its malfunction as an integral component of the channel complex. This study identifies the first AF-associated mutation in SCN3B, and suggests that mutations in SCN3B may be a new pathogenic cause of AF.     

Mutations in argininosuccinate synthetase mRNA of Japanese patients,causing classical citrullinemia.

Citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase. In order to characterize mutations in Japanese patients with classical citrullinemia, RNA isolated from 10 unrelated patients was reverse-transcribed, and cDNA amplified by PCR was cloned and sequenced. The 10 mutations identified included 6 missense mutations (A118T, A192V, R272C, G280R, R304W, and R363L), 2 mutations associated with an absence of an exon 7 or exon 13, 1 mutation with a deletion of the first 7 bp in exon 16 (which might be caused by abnormal splicing), and 1 mutation with an insertion of 37 bp within exons 15 and 16 in cDNA. The insertion mutation and the five missense mutations (R304W being excluded) are new mutations described in the present paper. These are in addition to 14 mutations (9 missense mutations, 4 mutations associated with an absence of an exon in mRNA, and 1 splicing mutation) that we identified previously in mainly American patients with neonatal citrullinemia. Two of these 20 mutations, a deletion of exon 13 sequence and a 7-bp deletion in exon 16, were common to Japanese and American populations from different ethnic backgrounds; however, other mutations were unique to each population. Furthermore, the presence of a frequent mutation--the exon 7 deletion mutation in mRNA, which accounts for 10 of 23 affected alleles--was demonstrated in Japanese citrullinemia. This differs from the situation in the United States, where there was far greater heterogeneity of mutations.     

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.     

Molecular Analysis and Phenotypic Study in 14 Chinese Families with Bietti Crystalline Dystrophy

Purpose

To investigate the clinical features and cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene mutations in 14 Chinese families with Bietti crystalline dystrophy (BCD).

Methods

Seventeen patients from 14 unrelated Chinese families with BCD were recruited for complete clinical ophthalmic examination and genetic study. The 11 exons of CYP4V2 were amplified from genomic DNA of all patients and their family members by polymerase chain reaction (PCR) and then sequenced. Exons of TIMP3 were also sequenced in BCD patient associated with choroidal neovascularization (CNV). One hundred and seventy unrelated healthy Chinese subjects were screened for mutations in CYP4V2.

Results

All 17 patients with BCD had mutations in CYP4V2; one of these mutations was novel (c.219T>A, p.F73L) and four other mutations had been reported. The p.F73L mutation was a commonly detected mutation in our study (seven out of 34 alleles), either in the homozygous state or in the heterozygous state. Among the patients, considerable phenotypic variability was detected, both within and between families. Screening of TIMP3 did not find any mutation in the BCD patient associated with CNV.

Conclusion

The novel CYP4V2 c.219T>A (p.F73L) mutation may be another recurrent mutation in Chinese patients with BCD. Our study expands the mutation spectrum of CYP4V2 and characterizes novel genotype–phenotype associations in Chinese patients with BCD.     

Mutations of the GATA4 and NKX2.5 genes in Chinese pediatric patients with non-familial congenital heart disease

A number of mutations in GATA4 and NKX2.5 have been identified to be causative for a subset of familial congenital heart defects (CHDs) and a small number of sporadic CHDs. In this study, we evaluated common GATA4 and NKX2.5 mutations in 135 Chinese pediatric patients with non-familial congenital heart defects. Two novel mutations in the coding region of GATA4 were identified, namely, 487C >T (Pro163Ser) in exon 1 in a child with tetralogy of Fallot and 1220C >A (Pro407Gln) in exon 6 in a pediatric patient with outlet membranous ventricular septal defect. We also found 848C >A (Pro283Gln) in exon 2 of the NKX2.5 gene in a pediatric patient with ventricular septal defect, patent ductus arteriosus and aortic isthmus stenosis. None of the mutations was detected in healthy control subjects (n = 114). This study suggests that GATA4 and NKX2.5 missense mutations may be associated with congenital heart defects in pediatric Chinese patients. Further clinical studies with large samples are warranted.     

A new ATP7B gene mutation with severe condition in two unrelated Iranian families with Wilson disease

Wilson disease is associated with a defect in copper metabolism and caused by different mutations in ATP7B gene. The aim of this study was to determine mutation frequency of ATP7B exons 8 and 14 in Wilson disease patients from the south of Iran. The exons 8 and 14 of ATP7B gene were analyzed in 65 unrelated Wilson disease patients by Denaturing High Performance Liquid Chromatography, and samples with abnormal peak profile were selected for direct DNA sequencing. Seven out of 65 (10.8%) patients had mutations at exon 14, including c.3061-1G>A in four and c.3207C>A in three patients. In addition, four different mutations were identified at exon 8 of six patients (9.2%). Three of these mutations have been previously reported, including c.2304delC in two patients, c.2293G>A and 2304dupC each in one patient. Furthermore, a novel mutation, c.2335T>G (p.Trp779Gly), was identified in two unrelated patients. The patients with this novel mutation demonstrated severe neuropsychiatric condition. All together, 13 out of 65 (20%) patients had mutations within exons 8 and 14. We also identified a lower frequency of the most common mutations of exons 8 and 14 in the southern Iranian population.     

Mutations in NEXN, a Z-disc gene, are associated with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM), the most common inherited cardiac disorder, is characterized by increased ventricular wall thickness that cannot be explained by underlying conditions, cadiomyocyte hypertrophy and disarray, and increased myocardial fibrosis. In as many as 50% of HCM cases, the genetic cause remains unknown, suggesting that more genes may be involved. Nexilin, encoded by NEXN, is a cardiac Z-disc protein recently identified as a crucial protein that functions to protect cardiac Z-discs from forces generated within the sarcomere. We screened NEXN in 121 unrelated HCM patients who did not carry any mutation in eight genes commonly mutated in myofilament disease. Two missense mutations, c.391C>G (p.Q131E) and c.835C>T (p.R279C), were identified in exons 5 and 8 of NEXN, respectively, in two probands. Each of the two mutations segregated with the HCM phenotype in the family and was absent in 384 control chromosomes. In silico analysis revealed that both of the mutations affect highly conserved amino acid residues, which are predicted to be functionally deleterious. Cellular transfection studies showed that the two mutations resulted in local accumulations of nexilin and that the expressed fragment of actin-binding domain containing p.Q131E completely lost the ability to bind F-actin in C2C12 cells. Coimmunoprecipitation assay indicated that the p.Q131E mutation decreased the binding of full-length NEXN to α-actin and abolished the interaction between the fragment of actin-binding domain and α-actin. Therefore, the mutations in NEXN that we describe here may further expand the knowledge of Z-disc genes in the pathogenesis of HCM.