Density functional theory studies on cytosine analogues for inducing double-proton transfer with guanine.

To induce double-proton transfer (DPT) with guanine in a biological environment, 12 cytosine analogues (Ca) were formed by atomic substitution. The DPT reactions in the Watson-Crick cytosine-guanine model complex (Ca0G) and 12 modified cytosine-guanine complexes (Ca1-12G) were investigated using density functional theory methods at the M06-2X/def2svp level. The intramolecular proton transfers within the analogues are not facile due to high energy barriers. The hydrogen bond lengths of the Ca1-12G complexes are shorter than those in the Ca0G complex, which are conducive to DPT reactions. The DPT energy barriers of Ca1-12G complexes are also lower than that of the Ca0G complex, in particular, the barriers in the Ca7G and Ca11G complexes were reduced to -1.33 and -2.02 kcal/mol, respectively, indicating they are significantly more prone to DPT reactions. The DPT equilibrium constants of Ca1-12G complexes range from 1.60 × 100 to 1.28 × 107, among which the equilibrium constants of Ca7G and Ca11G are over 1.0 × 105, so their DPT reactions may be adequate. The results demonstrate that those cytosine analogues, especially Ca7 and Ca11, are capable of inducing DPT with guanine, and then the guanine tautomer will form mismatches with thymine during DNA replication, which may provide new strategies for gene therapy.

A novel GJB1 mutation associated with X-linked Charcot-Marie-Tooth disease in a large Chinese family pedigree.

BACKGROUND: Charcot-Marie-Tooth (CMT) disease is a group of hereditary neuropathies with high phenotypic and genetic heterogeneity. In this study, we report a large family with X-linked CMT (CMTX) caused by a novel GJB1 mutation. METHODS: A family with the clinical diagnosis of CMTX was investigated. For mutation analysis, the coding region of GJB1 was sequenced using DNA from 15 family members. The identified GJB1 mutation was investigated by DHPLC in 120 normal controls. Mutation reanalysis was performed based on whole-exome sequencing (WES). Cell transfection studies were performed to characterize the function of the novel mutation. RESULTS: A missense mutation (c.605T>A) in GJB1 was detected in five patients and eight female carriers but not in two unaffected members of the family. The mutation was not found in 120 healthy controls and has not been previously reported. WES excluded other pathogenic mutations in the family. The pathogenicity of the mutation was confirmed by disrupting the membrane localization of the encoded proteins. CONCLUSION: Our findings demonstrate that a novel mutation (c.605T>A) in GJB1 is associated with CMTX and adds to the repertoire of GJB1 mutations related to CMTX.

A comprehensive multiplex PCR based exome-sequencing assay for rapid bloodspot confirmation of inborn errors of metabolism.

BACKGROUND: Tandem mass spectrometry (MS MS) and simple fluorometric assays are currently used in newborn screening programs to detect inborn errors of metabolism (IEM). The aim of the study was to evaluate the clinical utility of exome sequencing as a second tier screening method to assist clinical diagnosis of the newborn. METHODS: A novel PCR-exome amplification and re-sequencing (PEARS) assay was designed and used to detect mutations in 122 genes associated with 101 IEM. Newborn bloodspots positive by biochemical testing were analysed by PEARS assay to detect pathogenic mutations relevant to the IEM. RESULTS: In initial validation studies of genomic DNA samples, PEARS assay correctly detected 25 known mutations associated with 17 different IEM. Retrospective gene analysis of newborns with clinical phenylketonuria (PKU), identified compound heterozygote phenylalanine hydroxylase (PAH) gene mutations in eight of nine samples (89%). Prospective analysis of 211 bloodspots correctly identified the two true PKU samples, yielding positive and negative predictive values of 100%. Testing of 8 true positive MS MS samples correctly identified potentially pathogenic compound heterozygote genotypes in 2 cases of citrullinemia type 1 and one case each of methylmalonic acidemia, isobutyryl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase deficiency and glutaric acid type II and heterozygous genotypes in 2 cases of autosomal dominant methioninemia. Analysis of 11 of 12 false positive MS MS samples for other IEM identified heterozygous carriers in 8 cases for the relevant genes associated with the suspected IEM. In the remaining 3 cases, the test revealed compound heterozygote mutations in other metabolic genes not associated with the suspected IEM, indicating a misinterpretation of the original MS MS data. CONCLUSIONS: The PEARS assay has clinical utility as a rapid and cost effective second-tier test to assist the clinician to accurately diagnose newborns with a suspected IEM.

Low Expression of LEFTY1 in Placental Villi Is Associated with Early Unexplained Miscarriage.

OBJECTIVE: To investigate the function and underlying mechanism of transforming growth factor­beta (TGF-ß)/bone morphogenetic protein (BMP) signaling pathway in early unexplained miscarriage. STUDY DESIGN: Expression profiles of genes involved in TGF-ß/BMP signaling pathway were compared between placental villous tissue samples from 2 women with missed abortion and those from 2 women with induced abortion by microarray assay. The protein expression level of the most downregulated gene­LEFTY1­was further measured using western blotting in another 8 women with missed abortion and 7 women with induced abortion. RESULTS: A total of 24 genes showed differential expression level between the 2 groups. Their functions were further investigated, of which 6 of 13 upregulated genes were TGF-ß responsive genes. The most reduced gene is LEFTY1, an antagonist of TGF-ß ligand. The protein expression level of LEFTY1 was confirmed to show the same trend as microarray using western blotting. CONCLUSION: A reduced expression of LEFTY1 in women with missed abortion was identified as com-pared with women with induced abortion, which may result in a dysregulation of TGF-ß signaling and may be the underlying mechanism of missed abortion.

Whole exome sequencing identifies a novel missense FBN2 mutation co-segregating in a four-generation Chinese family with congenital contractural arachnodactyly.

BACKGROUND: Congenital contractural arachnodactyly (CCA) is an autosomal dominant rare genetic disease, estimated to be less than 1 in 10,000 worldwide. People with this condition often have permanently bent joints (contractures), like bent fingers and toes (camptodactyly). CASE PRESENTATION: In this study, we investigated the genetic aetiology of CCA in a four-generation Chinese family. The blood samples were collected from 22 living members of the family in the Yangquan County, Shanxi Province, China. Of those, eight individuals across 3 generations have CCA. Whole exome sequencing (WES) identified a missense mutation involving a T-to-G transition at position 3229 (c.3229 T > G) in exon 25 of the FBN2 gene, resulting in a Cys 1077 to Gly change (p.C1077G). This previously unreported mutation was found in all 8 affected individuals, but absent in 14 unaffected family members. SIFT/PolyPhen prediction and protein conservation analysis suggest that this novel mutation is pathogenic. Our study extended causative mutation spectrum of FBN2 gene in CCA patients. CONCLUSIONS: This study has identified a novel missense mutation in FBN2 gene (p.C1077G) resulting in CCA in a family of China.

A novel MSX1 intronic mutation associated with autosomal dominant non-syndromic oligodontia in a large Chinese family pedigree.

BACKGROUND: Tooth agenesis is a common developmental dental anomaly. The aim of the study was to identify the causal genetic mutation in a four-generation Chinese family affected with non-syndromic autosomal dominant tooth agenesis. METHODS: Genome-wide scanning was performed using the Illumina Linkage-12 array. Genotyping of short tandem repeat markers was used to finely map the causative locus. Haplotype analysis and Sanger sequencing was performed to precisely locate the position and nature of the gene defect. RESULTS: Clinical examination of the available 23 family members showed variable tooth agenesis in 10 subjects, ranging from oligodontia to mild hypodontia. Genome-wide scanning and haplotype analyses identified the 4p16.1-p16.3 region with a maximum multi-point LOD score of 3.50, which overlapped with the MSX1 gene. A single heterozygous point mutation IVS1-5 G>A in the MSX1 gene was exclusively detected in the 10 family members affected with tooth agenesis. Sequencing of MSX1 cDNA revealed that the intronic mutation did not affect the normal splicing pattern of the pre-mRNA. However, real-time qPCR analysis of lymphocyte RNA showed that the level of MSX1 mRNA was significantly decreased in individuals heterozygous for the mutation. CONCLUSIONS: We identified and characterized a novel intronic mutation in the MSX1 gene in a large Chinese pedigree, adding to the small repertoire of MSX1 mutations associated with autosomal dominant tooth agenesis. We hypothesize that the variable degree of tooth agenesis observed in each affected individual may be due to sub-optimal levels of MSX1 expression during critical stages tooth development.

Correlation between FMR1 expression and clinical phenotype in discordant dichorionic-diamniotic monozygotic twin sisters with the fragile X mutation.

BACKGROUND: The clinical phenotypes of females with fragile X full mutations vary drastically. Comparisons of discordant monozygotic twins provide opportunities to ascertain crucial factors that influence disease phenotype penetrance. OBJECTIVE: To identify crucial factors influencing the phenotypic expression of fragile X syndrome (FXS). METHODS AND RESULTS: We describe a pair of discordant monozygotic female twins (dichorionic-diamniotic, Di-Di) with full mutation. The degrees of their phenotypic discordance regarding physical, psychiatric and behavioural features were quantified in a series of neuropsychological tests that varied significantly. Their FMR1 expression levels and whole genome DNA methylation profiling in blood were similar. Their similar life experiences also suggested that environmental factors had limited influence. However, the skewed inactivation of the normal X chromosome in the hair roots of twin A, resulting in large reduction in FMR1 expression compared to that of twin B, could adequately explain their widely variable phenotypes. CONCLUSIONS: The sixfold variation in hair root FMR1 expression, which reflected FMRP (fragile X mental retardation protein) expression in the brain, accounted for the disparate phenotypes in IQ, cognition, and social capability between the twins. Additionally, considering the Di-Di type twinning and different CGG repeat sizes, CGG expansion should occur before splitting at day 3 of gestation.

[Effects of leflunomide on high glucose-induced podocyte cytoskeleton and its mechanism].

OBJECTIVE: To explore the effects of leflunomide active metabolite A771726 on high glucose-induced podocyte cytoskeleton and its possible signaling pathway. METHODS: The conditionally immortal human glomerular podocytes were divided into normal glucose (NG), mannitol (MA), high glucose (HG), high glucose with PDTC (pyrrolidine dithiocarbamate, a NF-κBp65 inhibitor) and high glucose with active leflunomide metabolite A771726 groups. Western blot was used to measure the ratio of p-NF-κBp65 to total NF-κBp65. And the protein and mRNA expressions of NF-κBp65, TRPC6 and nephrin were detected by Western blot and reverse transcription polymerase chain reaction (PCR). Immunofluorescence staining was used to detect the changes in the skeleton of podocyte. RESULTS: (1) Podocytes with high glucose could activate the NF-κBp65 signaling pathway. There was a significant increase of p-NF-κBp65 protein at 60 min versus 0 min (1.20 ± 0.04 vs 0.79 ± 0.02, P < 0.01). Little activation of the pathways was observed in groups NG and MA. The up-regulated protein expression of p-NF-κBp65 induced with high glucose was significantly inhibited by PDTC and A771726 (both P < 0.05). The difference of NF-κBp65 mRNA expression was not statistically significant between the groups (all P > 0.05). (2) High glucose-induced podocyte activated the NF-κBp65 signaling path. Its downstream TRPC6 mRNA and protein expression significantly increased than NG while nephrin became down-regulated more than NG. PDTC and A771726 inhibited the high expression of TRPC6 while the expression of nephrin was elevated (all P < 0.05). (3) Immunofluorescent assay of high glucose-induced podocyte cytoskeleton showed disorderly F-actin and a disappearance of tensile fiber after 72 h. CONCLUSION: Active leflunomide metabolite A771726 may protect podocytes through blocking the high glucose-induced signaling pathway of NF-κBp65.

Hutchinson-Gilford progeria syndrome accompanied by severe skeletal abnormalities in two Chinese siblings: two case reports.

INTRODUCTION: Hutchinson-Gilford progeria syndrome is a rare pediatric genetic syndrome with an incidence of one per eight million live births. The disorder is characterized by premature aging, generally leading to death due to myocardial infarction or stroke at approximately 13.4 years of age. The genetic diagnosis and special clinical manifestation in two Han Chinese siblings observed at our clinic for genetic counseling are described in this report. We screened the LMNA gene in these two siblings as well as in their unaffected parents. A homozygous mutation R527C was identified in the affected siblings, and both parents were heterozygous for this variant. CASE PRESENTATION: In case 1, the elder 10-year-old female sibling showed the classic physical and radiological changes of Hutchinson-Gilford progeria syndrome in addition to a considerable overlap with the phenotype of mandibuloacral dysplasia.In case 2, the younger male sibling had begun to show some early physical changes at age six months. CONCLUSION: The phenotypic findings in the patients we describe here widen the clinical spectrum of Hutchinson-Gilford progeria syndrome symptoms, providing further recognition of the phenotypic range of LMNA-associated diseases.

Mutation analysis in Chinese patients with Cornelia de Lange syndrome.

AIMS: Cornelia de Lange syndrome (CdLS) is a dominant multisystem developmental disorder and related to mutations of the NIPBL, SMC1A, and SMC3 genes. So far, there has been no report of a mutation analysis in Chinese patients with CdLS, while 12 cases have been clinically described. In the present study, we tried to search for pathogenic mutations of the NIPBL, SMC1A, and SMC3 genes in four patients with CdLS from four unrelated Chinese families. RESULTS: The mutational analysis of the NIPBL, SMC1A, and SMC3 genes by direct sequencing revealed a heterozygous splice-site mutation c.4321G>T(p.V1441L) at exon 20 of NIPBL in proband 2 and a novel heterozygous splice-site mutation c.6589+5G>C at intron 38 of NIPBL in proband 3, which was showed by reverse transcription polymerase chain reaction to generate both the full-length and an alternatively spliced transcript with an exon 38 deletion. CONCLUSIONS: This is the first report of the mutation analysis of NIPBL in China and our findings both expand the mutation spectrum of NIPBL and provide data for further understanding of the diverse and variable effects of NIPBL mutations.

A novel splicing mutation in COL1A1 gene caused type I osteogenesis imperfecta in a Chinese family.

Osteogenesis imperfect (OI) is a heritable connective tissue disorder with bone fragility as a cardinal manifestation, accompanied by short stature, dentinogenesis imperfecta, hyperlaxity of ligaments and skin, blue sclerae and hearing loss. Dominant form of OI is caused by mutations in the type I procollagen genes, COL1A1/A2. Here we identified a novel splicing mutation c.3207+1G>A (GenBank ID: JQ236861) in the COL1A1 gene that caused type I OI in a Chinese family. RNA splicing analysis proved that this mutation created a new splicing site at c.3200, and then led to frameshift. This result further enriched the mutation spectrum of type I procollagen genes.

Novel compound heterozygous mutation of MLYCD in a Chinese patient with malonic aciduria.

A 3-year-old Chinese boy presented with prominent clinical features of malonic aciduria, including developmental delay, short stature, brain abnormalities and massive excretion of malonic acid and methylmalonic acid. Molecular characterization by DNA sequencing analysis and multiplex ligation-dependent probe amplification of the MLYCD gene revealed a heterozygous mutation (c.920T>G, p.Leu307Arg) in the patient and his father and a heterozygous deletion comprising exon 1 in the patient and his mother. The missense mutation (c.920T>G) was not found in 100 healthy controls and has not been reported previously. Our findings expand the number of reported cases and add a novel entry to the repertoire of MLYCD mutations.

Novel multiplex primer extension and denaturing high-performance liquid chromatography for genotyping of the deafness gene mutations.

OBJECTIVE: To find a rapid and accurate genotyping method for specific non-syndromic hearing loss (NSHL)-causing gene mutations for disease diagnosis in different ethnic populations. METHODS: We performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) to simultaneously detect and genotype the 6 most common mutations in 180 patients with NSHL (GJB2-235delC, GJB2-299delAT, PDS-A2168G, PDS IVS7-2A>G, mtDNA-A1555G, and mtDNA-C1494T) in Chinese population. This method involved the amplification of the target sequence, followed by a purification step, a multiplex PE reaction, and DHPLC analysis performed on the Transgenomic Wave DNA fragment analysis system under fully-denaturing conditions. RESULTS: In a blind analysis, this technique successfully and accurately genotyped 100% of the samples simultaneously characterized by direct sequencing. CONCLUSION: Combination of PE and DHPLC is simple, rapid, accurate, and cost-effective for genotyping common disease-causing mutations, including substitutions, insertions, and deletions in NSHL, and may be successfully used in other genetic diseases.

A novel GPR143 splicing mutation in a Chinese family with X-linked congenital nystagmus.

PURPOSE: The purpose of the current research was to detect the underlying genetic defect in a Chinese family with X-linked congenital nystagmus and perform prenatal genetic diagnosis for their current pregnancy. METHODS: A common clinical examination and an ophthalmic evaluation were performed on the proband, one carrier, and one unaffected member. Mutation analysis of the G protein-coupled receptor 143 (GPR143) and four-point-one (4.1), ezrin, radixin, moesin (FERM) domain-containing 7 (FRMD7) genes was performed by direct sequencing of PCR-amplified exons in the proband. The detected GPR143 mutation was tested in all available family members and 200 normal controls by direct sequencing. RESULTS: Congenital nystagmus, obvious fundus hypopigmentation, and foveal hypoplasia were observed in the proband but not in the carriers or the unaffected members. A novel splicing mutation c.658+1 g>t not found in 200 unrelated controls was identified and co-segregated with X-linked ocular albinism (XLOA) in this family. The fetus (V:5) was hemizygous for this mutant allele. CONCLUSIONS: We identified a novel causative mutation of GPR143 in a five-generation Chinese family with XLOA. This expanded the mutation spectrum of GPR143 and provided data elucidating the diverse and variable effects of GPR143 mutations.

[Deletion and mutation analysis to FOXL2 in blepharophimosis-ptosis-epicanthus inversus syndrome].

OBJECTIVE: To perform genetic analysis in 5 patients with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and refine the genotype-phenotype correlation. METHODS: G-band karyotyping, fluorescent in situ hybridization (FISH), SNP array, PCR and sequencing techniques were performed to one patient with BPES and mental retardation and 4 only with BPES. RESULTS: Patient 1 with mental retardation carried a 9.4 Mb heterozygous deletion in chromosome 3q22.1-q23 including FOXL2 gene; Both patient 2 and 3 carried a c.704delG heterozygous mutation of FOXL2, while they were assigned to the different clinical type from those reported previously. Patient 3 was assigned to type II BPES; No mutation of FOXL2 was detected in patient 4 and 5. CONCLUSIONS: There might be the gene(s) responsible for mental retardation within chromosome 3q22.1-q23. It was indicated that the mutation c.704delG in FOXL2 led to a truncated protein is associated with both type I and II of BPES.

[Identification of the small supernumerary marker chromosomes in two patients with Turner syndrome].

OBJECTIVE: To identify the small supernumerary marker chromosomes (sSMC) and guide the genetic counseling and medical treatment in two patients with Turner syndrome. METHODS: High resolution GTG and C banding, SRY amplification by PCR and fluorescence in situ hybridization (FISH) on metaphase chromosomes were performed to the two patients. RESULTS: The karyotypes of the two patients were 45, X [29]/46,X, +mar[31] and 45,X[71]/46,X, +mar[29] respectively. SRY test indicated SRY-positive for patient 1, whose sSMC was originated from chromosome Y. The karyotype was confirmed as 45,X[29]/46,X,idic(Y)(q10)[31]. ish idic(Y)(q10)(RP11-115H13x2) (SRY+) by FISH. While in patient 2, the sSMC was originated from chromosome X, whose karyotype was determined as 45, X[71]/46,X, r(X)(p11.23q21)[29]. ish r(X) (p11.23q21)(AL591394.11xAC092268.3). CONCLUSION: Using cytogenetic and molecular cytogenetic analyses, we have identified the sSMCs in two patients with Turner syndrome, which was helpful to the clinical diagnosis and treatment.

[Molecular analysis of IDS gene and prenatal diagnosis in a Chinese family with mucopolysaccharidosis type II].

OBJECTIVE: Mucopolysaccharidosis type II (MPSII) is a lethal, X-linked recessive disorder caused by mutation of iduronate-2-sulfatase (IDS) gene. Up to now there is no really effective treatment for this disorder, therefore it is important to provide an accurate genetic diagnosis and prenatal diagnosis for the MPSII families. In this study, we identify the pathogenic mutation in a Chinese family with MPSII. METHOD: The 8 years old male proband from a Chinese family was clinically diagnosed with MPSII. There are other 4 patients with similar phenotypes in the family who died at 9, 11, 7 and 10 years of age, respectively. Mutation analysis was carried out by polymerase chain reaction and direct sequencing of all exons and exon/intron boundaries of IDS gene. Denaturing high performance liquid chromatography (DHPLC) analysis was performed to screen the unknown variations of IDS gene in 100 unrelated control males. RESULT: Two allelic variants of exon 5 (c.684A > G) and exon 6 (c.851C > T) and a nonsense mutation of exon 7 (c.892C > T) were detected in IDS gene of the proband. Heterozygous mutations c.684A > G, c.851C > T and c.892C > T were detected in both proband's mother and maternal grandmother. The unknown variations of c.684A > G and c.851C > T were not found in the 100 unrelated control males. The male fetus (IV11) inherited the same mutation of IDS gene as the proband. CONCLUSION: Mutation c.892C > T of IDS gene causes MPSII in this family and prenatal diagnosis in one affected fetus was achieved.

[Mutation screening of the dystrophin gene in 14 Chinese Duchenne/Becker muscular dystrophy patients without gross deletions].

OBJECTIVE: To search for the dystrophin gene mutations of Duchenne muscular dystrophy (DMD) patients without gross deletions, in order to offer accurate genetic counseling and prenatal diagnosis for those families. METHODS: All 79 exons of the dystrophin gene as well as its 5'-UTR and 3'-UTR of 14 Chinese DMD/Becker muscular dystrphy (BMD) patients without detectable gross deletions were screened by denaturing high performance liquid chromatography (DHPLC) and heteroduplex fragments were identified by subsequent sequencing. RESULTS: Seven causative point mutations, including two novel ones, were detected in 7 patients. Fourteen known polymorphisms and 7 unknown intronic variations were also detected. Five mothers of the patients were obligate carriers. CONCLUSION: DHPLC is an efficient way of identifying point mutations and the female carriers in DMD families.