Frequency of the ΔF508 deletion and G551D,R553X and G542X mutations in Yugoslav CF patients

Summary A study was undertaken to find the frequency of the F508 deletion and those of the G551D, R553X and G524X mutations among the mainly Slavic population of Serbia, Bosnia, Herzegovina, and Montenegro and compare the frequencies determined with those in other European populations. The F508 mutation was found to account for about 70% of CF genes in central Jugoslavia, where its frequency is significantly higher than elsewhere in Southern European populations.     

Identification of 28 novel mutations in the Bardet–Biedl syndrome genes: the burden of private mutations in an extensively heterogeneous disease

Bardet–Biedl syndrome (BBS), an emblematic disease in the rapidly evolving field of ciliopathies, is characterized by pleiotropic clinical features and extensive genetic heterogeneity. To date, 14 BBS genes have been identified, 3 of which have been found mutated only in a single BBS family each (BBS11/TRIM32, BBS13/MKS1 and BBS14/MKS4/NPHP6). Previous reports of systematic mutation detection in large cohorts of BBS families (n > 90) have dealt only with a single gene, or at most small subsets of the known BBS genes. Here we report extensive analysis of a cohort of 174 BBS families for 12/14 genes, leading to the identification of 28 novel mutations. Two pathogenic mutations in a single gene have been found in 117 families, and a single heterozygous mutation in 17 families (of which 8 involve the BBS1 recurrent mutation, M390R). We confirm that BBS1 and BBS10 are the most frequently mutated genes, followed by BBS12. No mutations have been found in BBS11/TRIM32, the identification of which as a BBS gene only relies on a single missense mutation in a single consanguineous family. While a third variant allele has been observed in a few families, they are in most cases missenses of uncertain pathogenicity, contrasting with the type of mutations observed as two alleles in a single gene. We discuss the various strategies for diagnostic mutation detection, including homozygosity mapping and targeted arrays for the detection of previously reported mutations.     

Spectrum of CFTR mutations in Mexican cystic fibrosis patients: identification of five novel mutations (W1098C, 846delT, P750L, 4160insGGGG and 297–1G→A)

We have analyzed 97 CF unrelated Mexican families for mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Our initial screening for 12 selected CFTR mutations led to mutation detection in 56.66% of the tested chromosomes. In patients with at least one unknown mutation after preliminary screening, an extensive analysis of the CFTR gene by single stranded conformation polymorphism (SSCP) or by multiplex heteroduplex (mHET) analysis was performed. A total of 34 different mutations representing 74.58% of the CF chromosomes were identified, including five novel CFTR mutations: W1098C, P750L, 846delT, 4160insGGGG and 297-1G-->A. The level of detection of the CF mutations in Mexico is still lower than that observed in other populations with a relatively low frequency of the deltaF508 mutation, mainly from southern Europe. The CFTR gene analysis described here clearly demonstrated the high heterogeneity of our CF population, which could be explained by the complex ethnic composition of the Mexican population, in particular by the strong impact of the genetic pool from southern European countries.     

Identification of missense, nonsense, and deletion mutations in the GRHPR gene in patients with primary hyperoxaluria type II (PH2)

Primary hyperoxaluria type II (PH2) is a rare disease characterized by the absence of an enzyme with glyoxylate reductase, hydroxypyruvate reductase, and D-glycerate dehydrogenase activities. The gene encoding this enzyme (GRHPR) has been characterized, and a single mutation has been detected in four PH2 patients. In this report, we have identified five novel mutations. One nonsense mutation (C295T) results in a premature stop codon at codon 99. A 4-bp deletion mutation has been found in the 5' consensus splice site of intron D, resulting in a predicted splicing error. Three missense mutations have been detected, including a missense transversion (T965G) in exon 9 (Met322Arg), a missense transition (G494A) in the putative co-factor binding site in exon 6 (Gly165Asp), and a substitution of an adenosine for a guanine in the 3' splice site of intron G. The functional consequences of the missense transversion and transition mutations have been investigated by transfection of cDNA encoding the mutated protein into COS cells. Cells transfected with either mutant construct have no enzymatic activity, a finding that is not significantly different from the control (empty) vector (P<0.05). These results further confirm that mutations in the GRHPR gene form the genetic basis of PH2. Ten of the 11 patients that we have genotyped are homozygous for one of the six mutations identified to date. Because of this high proportion of homozygotes, we have used microsatellite markers in close linkage with the GRHPR gene to investigate the possibility that the patients are the offspring of related individuals. Our data suggest that two thirds of our patients are the offspring of either closely or distantly related persons. Furthermore, genotyping has revealed the possible presence of a founder effect for the two most common mutations and the location of the gene near the marker D9S1874.     

Identification of novel Asian Indian and Japanese mutations causing β-thalassaemia in the Egyptian population

β-thalassaemia is a major health problem in Egypt. It has been estimated that of the 1.5 million live births, 1000 children with β-thalassaemia major are born annually. Although the available treatment has increased the life expectancy of patients, it is still unsatisfactory and represents a significant drain on the country’s resources. National screening and prenatal diagnosis programmes can be provided in Egypt once the spectrum of β-thalassaemia mutations has been identified within the Egyptian population. We have examined 16 DNA samples with 21 β-thalassaemia mutations that remained unidentified in a study of 54 patients reported by Rady and colleagues in 1996. Using the polymerase chain reaction and single strand conformation analysis we identified the following changes: frameshift (FS) codon (CD) 8/9 (+G), 4 FS CD 29 (–G) and 2 novel mutations in exon I (15 CD 22 A-C and 1 FS CD 28 –C). In addition, a silent, probably polymorphic mutation, CD 17 G-A was present in all chromosomes. Received: 19 August 1996 / Revised: 21 September 1996     

Identification of four novel connexin 26 mutations in non-syndromic deaf patients: genotype–phenotype analysis in moderate cases

This paper presents a mutation as well as a genotype–phenotype analysis of the GJB2 and GJB6 genes in 476 samples from non-syndromic unrelated Argentinean deaf patients (104 familial and 372 sporadic cases). Most of them were of prelingual onset (82 %) and 27 % were cochlear implanted. Variation of sequences was detected in 171 of the 474 patients (36 %). Overall, 43 different sequence variations were identified in GJB2 and GJB6. Four of them are reported for the first time in GJB2: c.233dupG, p.Ala78Ser, p.Val190Asp and p.Cys211Tyr. Mutations in GJB6 were detected in 3 % of patients [nine del(GJB6-D13S1830) and three del(GJB6-D13S1854)]. Of the 43 different variations identified in GJB2, 6 were polymorphisms and of the others, 10 (27 %) were truncating and 27 (73 %) were nontruncating. Patients with two truncating mutations had significantly worse hearing impairment than all other groups. Moderate phenotypes were observed in a group of patients carrying biallelic mutations (23 %). This work shows the high prevalence of GJB2 mutations in the Argentinean population and presents an analysis of moderate phenotypes in our cohort.     

Significance of each of three missense mutations, G484A, G667A, and G808A, present in an inactive allele of the human Lewis gene (FUT3) for α(1,3/1,4)fucosyltransferase inactivation

Recently, we found three novel missense mutations, G484A (Asp162Asn), G667A (Gly223Arg), and G808A (Val270Met), present in a Lewis-negative allele (le484,667,808) from an African (Xhosa) population. To define the relative contribution of each of the three mutations in the le484,667,808 allele for inactivation of the FUT3-encoded enzyme, we made chimeric FUT3 containing each of the three mutations. A transient expression study indicated that COS7 cells transfected with the FUT3 construct containing the G484A mutation expressed the Lewis antigen and had about 20% enzyme activity as compared with COS7 cells transfected with the wild type FUT3 allele, whereas COS7 cells transfected with the FUT3 construct containing either the G667A mutation or the G808A mutation did not express the Lewis antigen and showed no detectable (1,3/1,4)fucosyltransferase activity. These results suggest that the G667A and/or the G808A missense mutations of FUT3 alleles are responsible for the inactivation of the FUT3-encoded enzyme.     

Identification of novel SNPs in SYK gene of breast cancer patients: computational analysis of SNPs in the 5′UTR

Spleen tyrosine kinase (SYK) is a non receptor type tyrosine kinase and a known candidate tumor suppressor gene in breast carcinoma. Loss of Syk is associated with breast cancer invasion and increased cell mortality. The main goal of our study was to detect germ-line polymorphisms in SYK gene in breast cancer affected females of Pakistani origin, in order to understand the genetic basis of complex human breast cancer. Seven novel SYK gene SNPs were identified in breast cancer patients. Among these, three were identified in intronic region, one at the 5'splice donor site (5'SD) and three in 5'untranslated region (5'UTR) of SYK gene. Mutations at the 5'SD and at 5'UTR can be crucial and could be responsible for generation of mutated Syk protein. In silico analysis of the 5'UTR variations revealed that one of the mutations was responsible for generation of a more stable structure of 5'UTR. Such a change in pre-mRNA could potentially down regulate SYK expression. These findings add to the growing literature implicating dysfunctional SYK gene as a contributor to human breast cancer, and suggest that therapies targeting its molecular pathways could provide effective means of treating/preventing breast cancer.     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

Computational investigation on the effects of H50Q and G51D mutations on the α-Synuclein aggregation propensity

The aggregation of α-synuclein is linked directly to the histopathology of Parkinson’s disease (PD). However, several missense mutations present in the α-synuclein gene (SNCA) have been known to be associated with PD. Several studies have highlighted the effect of SNCA mutations on the α-synuclein aggregation, but their pathological roles are not completely established. In this study, we have focused on the effects of the recently discovered α-synuclein missense mutants (H50Q and G51D) on the aggregation using computational approaches. We performed all atom molecular dynamics (MD) simulation on these mutants and compared their conformational dynamics with Wild-Type (WT) α-synuclein. We noticed the solvent accessible surface area (SASA), radius of gyration, atomic fluctuations, and beta strand content to be higher in H50Q than G51D and WT. Using PDBSum online server; we analyzed the inter-molecular interactions that drive the association of monomeric units of H50Q, WT, and G51D in forming the respective homo-dimer. We noticed the interface area, number of interacting residues and binding free energy to be higher for H50Q homo-dimer than the WT and G51D homo-dimers. Our findings in this study suggest that in comparison to WT and G51D, H50Q mutation to have a positive effect on increasing the α-synuclein aggregation propensity. Hence, we see that H50Q and G51D mutation show conflicting effect on the aggregation propensity of α-synuclein.     

Identification and characterization of the carboxy-terminal region of Sip-1, a novel autoantigen in Behçet's disease

Given the lack of a serological test specific for Behçet's disease, its diagnosis rests upon clinical criteria. The clinical diagnosis is nevertheless difficult because the disease manifestations vary widely, especially at the onset of disease. The aim of this study was to identify molecules specifically recognized by serum autoantibodies in patients with Behçet's disease and to evaluate their diagnostic value. We screened a cDNA library from human microvascular endothelial cells with serum IgG from two patients with Behçet's disease and isolated a reactive clone specific to the carboxy-terminal subunit of Sip1 (Sip1 C-ter). Using ELISA, we measured IgG, IgM and IgA specific to Sip1 C-ter in patients with various autoimmune diseases characterized by the presence of serum anti-endothelial cell antibodies, such as Behçet's disease, systemic lupus erythematosus, systemic sclerosis and various forms of primary vasculitis, as well as in patients with diseases that share clinical features with Behçet's disease, such as inflammatory bowel disease and uveitis. IgM immunoreactivity to Sip1 C-ter was significantly higher in patients with Behçet's disease and in patients with primary vasculitis than in the other groups of patients and healthy subjects tested (P < 10^-4 by Mann-Whitney test). ELISA detected IgG specific to Sip1 C-ter in sera from 11/56 (20%) patients with Behçet's disease, IgM in 23/56 (41%) and IgA in 9/54 (17%). No sera from patients with systemic lupus erythematosus, systemic sclerosis, inflammatory bowel disease, uveitis or healthy subjects but 45% of sera from patients with primary vasculitis contained IgM specific to Sip1 C-ter. Serum levels of soluble E-selectin, a marker of endothelial activation and inflammation, correlated with levels of serum IgM anti Sip-1 C-ter in patients with Behçet's disease (r = 0.36, P = 0.023). In conclusion, Sip1 C-ter is a novel autoantigen in Behçet's disease. IgM specific to Sip1 C-ter might be useful in clinical practice as an immunological marker of endothelial dysfunction in vasculitis.     

Effects of mutations in genesfadR,fabB, fadE,andenvC ofEscherichia coli on the action of the lysis gene of bacteriophageϕX174

The lytic effect of the expression of the cloned geneE of bacteriophage X174 inEscherichia coli is considerably amplified by a mutation in thefadR gene, which primarily affects the regulation of fatty acid degradation. In contrast, reduction of the fluidity of the cell membranes by use of thefabB andfadE mutations, which interfere with the synthesis and the oxidation of unsaturated fatty acids, severely inhibits the action of the X174 lysis gene product. A chain-forming mutant carrying a pleiotropic mutation in theenvC locus is also refractory to the X174 lysis protein. As shown by reversion and complementation of theenvC mutatation, a defect in at least one additional gene (rle) is involved in the generation of this refractoriness.     

Identification of novel mutations in the ABCA12 gene,c.1857delA and c.5653–5655delTAT,causing harlequin ichthyosis

Harlequin ichthyosis (HI) is a severe autosomal recessive developmental disorder of the skin that is frequently but not always fatal in the first few days of life. In HI, mutations in both ABCA12 gene alleles must have a severe impact on protein function and most mutations are truncating. The presence of at least one nontruncating mutation (predicting a residual protein function) usually causes a less severe congenital ichthyosis (lamellar ichthyosis or congenital ichthyosiform erythroderma). Here we report on a girl with severe HI diagnosed by prenatal ultrasound at 33 5/7 week gestation. Ultrasound findings included ectropion, eclabium, deformed nose, hands and feet, joint contractures, hyperechogenic amniotic fluid and polyhydramnion. After birth, palliative treatment was provided and she died on her first day of life. Sequence analysis of the ABCA12 gene identified two novel mutations, c.1857delA (predicting p.Lys619*) in exon 15 and c.5653–5655delTAT (predicting p.1885delTyr) in exon 37, each in heterozygous state. The c.5653–5655delTAT mutation is not truncating, but the deleted tyrosine at position 1885 is perfectly conserved among vertebrates and molecular studies evaluated the mutation as probably disease causing and damaging.     

CFTR gene mutations – including three novel nucleotide substitutions – and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease

In order to investigate the incidence of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and unclassified variants in chronic pulmonary disease in children and adults, we studied 20 patients with asthma, 19 with disseminated bronchiectasis (DB) of unknown aetiology, and 12 patients with chronic obstructive pulmonary disease (COPD), and compared the results to 52 subjects from the general Greek population. Analysis of the whole coding region of the CFTR gene and its flanking intronic regions revealed that the proportion of CFTR mutations was 45% in asthma (P<0.05), 26.3% in DB (P>0.05), 16.7% in COPD (P>0.05), compared to 15.4% in the general population. Seventeen different molecular defects involved in disease predisposition were identified in 16 patients. Three potentially disease-causing mutations, T388 M, M1R and V11I, are novel, found so far only in three asthma patients. The hyperactive M470 allele was found more frequently in COPD patients (frequency 70.8%, P<0.01) than in the controls. The study of the TGmTnM470 V polyvariant CFTR allele revealed the presence of CFTR function-modulating haplotypes TG13/T5/M470, TG11/T5/M470, TG12/T5/V470 and TG12/T7, combined with M470 or V470, in six asthma patients, four DB patients (P<0.01), and two COPD patients (P<0.05). These results confirm the involvement of the CFTR gene in asthma, DB and possibly in COPD.     

A point mutation,R59G,within the HMG-SRY box in a female 45,X/46,X,psu dic(Y)(pter→q11::q11→pter)

We report a molecular and cytogenetic investigation of a psu dic(Yp) chromosome identified in blood and ovarian tissue from a female with mosaic karyotype 45,X/46,X,+ psu dic(Yp). FISH analysis showed that the psu dic(Yp) has two copies of the short arm, two centromeres and two copies of the proximal long arm. PCR analysis also confirmed the presence of the SRY gene and the Y centromere, and also confirmed the deletion of the Y-heterochromatic region. Because of the possibility of a mutation, a fragment of 609 bp of the SRY gene was sequenced from independent PCR products. The analysis of the sequence indicated the presence of two different copies of the gene: one presented a point mutation, R59G, within the HMG-box; the other had a sequence identical to that already published. Both sequences were found at a proportion of 1:1. The absence of a 46,XY cell line suggests that the rearrangement took place during gametogenesis or during the first division after fertilization. Also, the existence of different sequences of the SRYgene in the same Y chromosome suggests that the formation of the dicentric took place prior to the mutation of the SRY gene. To our knowledge, this is the first time that a mutation has been described in codon 59 within the HMG- SRY box, and also the first case of a psu dic(Yp) chromosome that displays two different copies of the SRY gene.     

Women heterozygous for deficiency of the (p21 → pter) region of the X chromosome are fertile

Summary A woman balanced carrier of a X/15 translocation gave birth to a balanced infertile son and three unbalanced Xp- fertile daughters. This family and the other eleven cases of Xp- fertile women found in the literature demonstrate that loss of the p21 pter region of the X chromosome is compatible with fertility, probably because it leaves on Xp the region which is never inactivated.     

Identification,characterization, and developmental expression of a novel α2 → 8-KDN-transferase which terminates elongation of α2 → 8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2–5mm Mg²⁺ or Mn²⁺. Expression of KDN-transferase was developmentally regulated in parallel with expression of the 2 8-polysialytransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in capping of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of 2 8-polysialylation in glycoproteins.Abbreviations KDN 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid or naturally occurring deaminated neuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ge N-glycolylneuraminic acid - CMP-KDN cytidine 5-(3-deoxy-d-glycero-d-galacto-2-nonulosonic phosphate) or cytidine 5-KDN phosphate - CMP-NeuAc cytidine 5-Neu5Ac phosphate; oligo-polySia, oligo- and/or polysialic acid - PSGP rainbow trout egg polysialoglycoprotein comprising 2 8-linked oligo- polyNeu5Gc - PSGP (low Sia) a precursor of PSGP present at early stages of oogenesis which contains mostly the disialyl group, Sia2 8Sia2 6- - *K-PSGP [¹⁴C]KDN-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]KDN with the immature cortical vesicle fraction P1 containing KDN-transferase - *A-PSGP [¹⁴C]Neu5Ac-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]Neu5Ac with the P1 fraction - A-*K-PSGP andK-*K-PSGP the products obtained after incubating *K-PSGP with P1 fraction and unlabelled CMP-Neu5Ac or CMP-KDN, respectively - *K-PSGP _cho ,A-*K-PSGP _cho , andK-*K-PSGP _cho mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of *K-PSGP,A-*K-PSGP, and K-*K-PSGP, respectively - *A-PSGP _cho a mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of [¹⁴C]Neu5Ac-labelled PSGP - Endo-N endo-N-acylneuraminidase - DP degree of polymerization - GLC gas-liquid chromatography - HPLC high performance liquid chromatography - TLC thin layer chromatography