Novel mutations in the STK11 gene in Thai patients with Peutz-Jeghers syndrome

Peutz-Jeghers syndrome (PJS), a rare autosomal dominant inherited disorder, is characterized by hamartomatous gastrointestinal polyps and mucocutaneous pigmentation. Patients with this syndrome have a predisposition to a variety of cancers in multiple organs. Mutations in the serine/threonine kinase 11 (STK11) gene have been identified as a major cause of PJS. Here we present the clinical and molecular findings of two unrelated Thai individuals with PJS. Mutation analysis by Polymerase Chain Reaction-sequencing of the entire coding region of STK11 revealed two potentially pathogenic mutations. One harbored a single nucleotide deletion (c.182delG) in exon 1 resulting in a frameshift leading to premature termination at codon 63 (p.Gly61AlafsX63). The other carried an in-frame 9-base-pair (bp) deletion in exon 7, c.907_915del9 (p.Ile303_Gln305del). Both deletions were de novo and have never been previously described. This study has expanded the genotypic spectrum of the STK11 gene.     

Identification of 123 previously unreported mutations in the F8 gene of Iranian patients with haemophilia A

In Haemophilia A (HA), the deficiency in coagulation factor VIII is caused by mutations in the F8 gene. In the past, HA carrier detection in Iran used to be carried out by tracking polymorphic DNA markers - a technical strategy with poor efficacy and accuracy. For some 10 years, however, mutations have been identified by direct DNA sequencing at the Iranian Comprehensive Haemophilia Care Centre (ICHCC), resulting in the detection of 580 different mutations and accurate carrier detection. The aim of this study was to characterize and report the unreported mutations not recorded in the F8 HAMSTeRS database and HGMD, which we have detected amongst all the mutations hitherto identified. After excluding introns 1 and 22 inversions, direct DNA sequencing was used to detect mutations among our patients. These were then confirmed in another affected relative or obligate carrier. Severe cases of HA, where no mutation could be identified, were further investigated by the MLPA method. The new, unreported mutations identified include: 51 missense, 15 nonsense, 45 frame-shifts, 11 splice-site, 1 duplications. We report a large spectrum of mutations identified in the course of the past 10 years at the ICHCC, which offers this service to all patients from regions throughout Iran. Aside from the common introns 1 and 22 inversions, this work demonstrates a high degree of heterogeneity in F8 mutations. The establishment of a comprehensive Iranian HA database will improve the care and genetic counselling of Iranian HA families.     

F508del CFTR gene mutation in patients with allergic bronchopulmonary aspergillosis

Objective: The F508del mutation occurs in approximately 3.5% of Caucasian population of Northern Europe. Heterozygotes have increased risk for asthma and reduced pulmonary function. Allergic bronchopulmonary aspergillosis (ABPA) is more common in patients with cystic fibrosis (CF). We aimed to establish the frequency of F508del mutation in adult patients with ABPA. Methods: A retrospective matched case-control study of CF genotyped patients with ABPA seen at the National Aspergillosis Centre was undertaken. Key data were collected retrospectively from medical records, including respiratory comorbidities, total IgE, Aspergillus IgG and IgE, and immunoglobulins. Cystic fibrosis transmembrane regulator (CFTR) gene mutation analysis included multiplex PCR and sequencing. Results: From a cohort of 189 ABPA patients, 156 were screened for common mutations and variants in the CFTR gene. Eighteen were heterozygous for at least one CFTR mutation; 12 (7.7%) were heterozygous for the F508del, notably; 3 were heterozygous for the intron 8 5T variant; and 1 for an intronic variant of uncertain significance, c.3139 + 18C>T. Eight (67%) had asthma, 7 (58%) had CT-defined bronchiectasis, 4 (33%) hypergammaglobulinemia (>16 g/L), 3 (25%) sinusitis and 1 (8%) chronic pulmonary aspergillosis. Eight (67%) had elevated Aspergillus IgG antibodies (42–98 mg/L), and 8 (67%) had total IgE above 1,000 KIU/L. Two individuals heterozygous for the F508del mutation and the TG12T5 variant were diagnosed with CF, leading to a de novo CF discovery rate of 1.3%. Conclusions: In our ABPA patient cohort, the presence of the delta F508 mutation was higher than that seen in general population. Genetic counseling for CFTR genotyping might be appropriate for these patients.     

A focus on dominant negative variants in a series of 170 heterozygous FXI-deficient patients

Introduction

Dominant-negative effects have been described for 10 F11 variants in the literature.

Aim

The current study aimed at identifying putative dominant-negative F11 variants.

Material and methods

This research consisted in a retrospective analysis of routine laboratory data.

Results

In a series of 170 patients with moderate/mild factor XI (FXI) deficiencies, we identified heterozygous carriers of previously reported dominant-negative variants (p.Ser243Phe, p.Cys416Tyr, and p.Gly418Val) with FXI activities inconsistent with a dominant-negative effect. Our findings also do not support a dominant-negative effect of p.Gly418Ala. We also identified a set of patients carrying heterozygous variants, among which five out of 11 are novel, with FXI activities suggesting a dominant-negative effect (p.His53Tyr, p.Cys110Gly, p.Cys140Tyr, p.Glu245Lys, p.Trp246Cys, p.Glu315Lys, p.Ile421Thr, p.Trp425Cys, p.Glu565Lys, p.Thr593Met, and p.Trp617Ter). However, for all but two of these variants, individuals with close to half normal FXI coagulant activity (FXI:C) were identified, indicating an inconstant dominant effect.

Conclusion

Our data show that for some F11 variants recognized has having dominant-negative effects, such effects actually do not occur in many individuals. The present data suggest that for these patients, the intracellular quality control mechanisms eliminate the variant monomeric polypeptide before homodimer assembly, thereby allowing only the wild-type homodimer to assemble and resulting in half normal activities. In contrast, in patients with markedly decreased activities, some mutant polypeptides might escape this first quality control. In turn, assembly of heterodimeric molecules as well as mutant homodimers would result in activities closer to 1:4 of FXI:C normal range.     

Molecular spectrum of alpha-thalassemia in the Iranian population of Hormozgan: three novel point mutation defects

We describe the molecular spectrum of alpha-thalassemia mutations in a population sample of newborns in the South-Iranian province of Hormozgan. Out of 660 randomly collected blood samples 218 (33%) had visibly elevated Hb Bart's. DNA was extracted from 78 samples out of this selection (n=156), of which 114 alleles were found to carry an alpha-thalassemia defect. Besides the common -alpha3.7 (79.1%), -alpha4.2 (1.7%), and alpha-5nt alpha alleles (4.3%), three novel nondeletional alpha-thalassemia mutations were found; the alpha2 cd19 (-G) frameshift mutation (12.2%), the alpha1 IVS1-148(A-->G) (0.9%) affecting the splice acceptor site consensus sequence and the cd14 (TGG-->TAG) (0.9%), which creates a premature stop codon in the first exon of the alpha1-gene. A fourth mutation in the alpha1-gene, the IVS1-38 (C-->T) (0.9%) of undetermined effect, was found in an individual heterozygous for the alpha2 cd19(-G) mutation.     

Thirteen novel mutations in the factor VIII gene in the Nijmegen haemophilia A patient population

The development of neutralising antibodies to factor VIII (FVIII) is a major complication of haemophilia A (HA) therapy. We aimed to construct an individual risk profile for the development of inhibitors in HA and started by screening for the causative mutation in our HA patient population. A total of 109 patients and 28 carriers were screened. The analysis revealed 38 different mutations in the FVIII gene, of which 13 have not been described on the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS). Twenty-five mutations have been reported previously and all except two had a similar phenotype to what has been described. Three novel mutations were associated with severe HA: one non-missense mutation, a small insertion in the A2 domain, and two missense mutations, a H256R mutation in the A1 domain and a L2025P substitution in the C1 domain. One novel mutation, Y156C, was associated with moderate HA. Nine novel mutations caused mild HA. The P130R, D167E and V278M mutations are located in the A1 domain. R439C, Y511H, A544G and Q645H in the A2 domain, L1758F in the A3 domain and a S2157R mutation in the C1 domain. In conclusion, the genotypic profile of our HA population was not different from others described and is suitable to study inhibitor formation.     

Three novel point mutations causing haemophilia A

Haemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII gene. In our efforts to elucidate molecular defects in the haemophilia A patients from the Republic of Macedonia, we employed nonradioactive single-strand conformation polymorphism analysis followed by direct sequencing, for identifying point mutations in the factor VIII gene. In the present study we report the detection of three novel missense mutations: Met19 --> Arg; Ala78 --> Pro and Cys2174 --> Gly, all causing haemophilia A.     

Two novel factor VII gene mutations in a Chinese family with factor VII deficiency

We report two novel factor VII (FVII) gene mutations in a Chinese family with FVII deficiency. The proband, a 55-year-old woman. was incidentally found to have right shoulder arthritis consistent with chronic haemophilic arthropathy. FVII studies showed a FVII activity of 0.02 iu/ml and a FVII antigen of 49%. Molecular analysis showed a double heterozygous state, with an exon 4 nonsense mutation (C6003-->A; Cys61-->Term) and an exon 8 missense mutation (T10902-->G; Cys329-->Gly) that disrupted a Cys310/Cys329 disulphide bond. The genotypes and phenotypes were correlated in the patient's daughters. Two daughters were heterozygous for the Cys61-->Term mutation and showed a type 1 FVII gene mutation phenotype consistent with a nonsense mutation. One daughter was heterozygous for the Csy329-->Gly mutation and showed a type 2 mutation phenotype consistent with a missense mutation. These are the first reported FVII gene mutations in the Chinese people.     

Spectrum of mutations in Albanian patients with haemophilia A: identification of ten novel mutations in the factor VIII gene

Genetic analysis was carried out in 37 Albanian patients with haemophilia A. The factor VIII intron 22 inversion was detected only in 2/19 (10.5%) apparently unrelated patients with severe haemophilia A, while the intron 1 inversion was absent. A total of 19 different gene mutations were identified. Ten mutations were novel: four null mutations in severe haemophilia A patients (Gln1090X, Cys1832X, 2374delT, 5676insT) and six missense mutations (five in severe haemophilia A) (Ile76Thr, Leu299Pro, Asp525Glu, Cys692Tyr, His1755Leu and Trp1835Cys). None of these novel mutations occurred at CpG hotspots. These results further emphasize the extreme heterogeneity of the molecular basis of haemophilia A. The low prevalence of intron 22 inversion in Albanian patients with severe haemophilia A should be addressed by further studies.     

Frequency of HFE gene mutations in Iranian beta-thalassaemia minor patients

OBJECTIVES: Beta-thalassaemia minor (BTM) alone does not lead to iron overload, however, some gene modifiers and acquired causes are reported. When it is inherited together with a mutation in the HFE (HLA-H) gene associated with hereditary haemochromatosis, iron overload may ensue. To analyse the relationship between iron status and HFE mutations in Iranian BTM patients, we compared the frequency of the C282Y and H63D HFE mutations and ferritin level in a group of BTM patients from the National Thalassaemia Transfusion and Care Centre with that of healthy individuals. PATIENTS AND METHODS: Ninety-three (56 females) documented BTM cases and 104 (54 females) controls were enrolled in the study. Serum ferritin level was measured in all subjects by immuno-radiometric assay and HFE genotypes were determined using restriction fragment length polymorphism analysis of PCR-amplified HFE gene fragment. RESULTS: Eighteen (19.4%) BTM patients vs. 12 (11.5%) controls were H63D heterozygotes, while there were three (3.2%) cases and three (2.9%) controls with H63D homozygosity. All three C282Y mutations were found in BMT patients with one of them being a compound heterozygote. A significant difference was observed in the total number of HFE mutations in favour of BTM patients over the controls (P < 0.05, OR = 2.064). The H63D and C282Y allele frequencies were 12.9 and 1.61 in patients and 8.65 and 0 in controls, respectively. The mean ferritin level in cases with HFE mutations showed no significant difference from that of the patients without mutations (P > 0.05). CONCLUSIONS: Our results suggest that HFE mutations C282Y and H63D are more frequent in Iranian BTM patients than in the normal population, causing no significant changes in serum ferritin level.     

Detection of Ki-ras gene point mutations in bile specimens for the differential diagnosis of malignant and benign biliary strictures

BACKGROUND AND AIM: The present study was undertaken to determine if detection of Ki-ras gene point mutations in bile specimens could differentiate between benign and malignant biliary strictures. PATIENTS: Bile specimens were obtained from 117 patients exhibiting a stricture of the main bile duct, the nature of which was assessed by cholangiography, histology, and follow up. METHODS: DNA from frozen bile specimens was extracted, amplified, and tested for codon 12 point mutations of Ki-ras gene using sequence specific oligonucleotide hybridisation and mutant allele specific amplification. RESULTS: DNA amplification was successful in 110/117 bile specimens (94%). Detection of Ki-ras gene mutations in bile specimens was positive in 24.4% (22/90) of patients with malignant strictures, in 31.4% (22/70) when only primary malignant tumours were considered, and in 4% (1/25) of patients with benign strictures. Of the 49 patients with histological specimens obtained before surgery, the sensitivity of histology, Ki-ras mutation analysis, and combined methods was 59.2%, 28.6%, and 73.5% respectively. CONCLUSIONS: Our study showed that Ki-ras mutations may be detected in about one third of bile specimens from patients with primary tumours invading the main bile duct. Detection of such mutations appears to be specific and may help to differentiate between benign and malignant biliary strictures.     

PK-LR gene mutations in pyruvate kinase deficient Portuguese patients

In nine unrelated Portuguese patients with pyruvate kinase (PK) deficient anaemia, whose symptoms ranged from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring multiple transfusions, the PK-LR gene mutations were identified and correlated with their phenotypes. Five different mutations were identified, three of them for the first time: a missense mutation 1670G --> C on exon 12 and two 5' splice donor site (GT) mutations on intron 8 [IVS8(+2)T --> G] and intron 10 [IVS10(+1)G --> C]. Two previously described missense mutations, 1456C --> T and 993C --> A, were also found. The genotype/phenotype correlation showed that patients with two missense mutations or with a missense mutation and a splicing mutation had a mild haemolytic anaemia. The three patients with severe anaemia, who were transfusion dependent until splenectomy, were homozygous for the splicing site mutations IVS10(+1)G --> C or IVS8(+2)T --> G.     

Characterization of two novel splice site mutations in human factor VII gene causing severe plasma factor VII deficiency and bleeding diathesis

The molecular basis of severe type I factor (F)VII deficiency was investigated in two Algerian patients. One patient, a 13-year-old-girl who has suffered from severe bleeding since birth, was homozygous for a 7-bp deletion (nt 7774-7780) and a 251-bp insertion (nt 7773-7781) of mitochondrial origin, in IVS 4 acceptor splice site. The other patient, an infant who died from massive intracranial haemorrhage, was homozygous for a transversion in the IVS 7 donor splice site (T9726+2-->G) and a missense mutation in exon 8 (G10588-->A; Arg224-->Gln). In both cases, the deleterious mutations are probably the splice site junction abnormalities impairing mRNA processing. These three lesions have not yet been reported.     

Triose phosphate isomerase deficiency associated with two novel mutations in TPI gene

We report the clinical, haematological and molecular characteristics of two triose phosphate isomerase deficient patients affected by haemolytic anaemia and neuromuscular impairment. The sequence of complete TPI gene showed the presence of two previously undescribed mutations: c.722 T>C (Phe240Ser) and c.28 insG; each of the two unrelated patients showed the new mutation in compound heterozygosity with the most common variant Glu104Asp. The association of Glu104Asp with c.28 insG resulted in a very severe clinical pattern.