Characteristic perforin gene mutations of haemophagocytic lymphohistiocytosis patients in Japan

Perforin gene (PRF1) mutations appear to occur in about 30% of patients with haemophagocytic lymphohistiocytosis (HLH). We tested perforin expression and gene mutations in 14 HLH patients and six patients with Epstein-Barr virus-associated HLH (EBV-HLH) in Japan. Five of the 14 HLH patients had perforin abnormalities. The presence of PRF1 genetic abnormality correlated well with the lack of perforin expression as determined by flow cytometry. Sequencing showed that four patients had a compound heterozygous mutation while the fifth patient had a homozygous mutation. Three of the mutations we detected were novel. In contrast, none of the six EBV-HLH patients showed perforin abnormalities. Our data, combined with the PRF1 mutations in three previously reported Japanese patients, suggest that the 1090-1091delCT and 207delC mutations of the perforin gene are frequently present in Japanese HLH patients (62.5% and 37.5% respectively). Examination of the geographical origins of the ancestors in the perforin-mutant HLH patients revealed that they mostly came from the Western part of Japan, suggesting that the present-day cases may largely derive from a common ancestor.     

Identification of a novel mutation in the PAX9 gene in a family affected by oligodontia and other dental anomalies

The objective of the present work was to study the phenotype and the genotype of three generations of a family affected by oligodontia and other dental anomalies. These family members also presented systemic conditions such as hypercholesterolemia, hypothyroidism, diabetes mellitus, scoliosis, and congenital cardiovascular anomalies. Clinical evaluation, panoramic radiographs, and anamnestic data were used for dental analysis. DNA extraction was carried out from gum samples or buccal swabs. A mutation was identified in six subjects across three generations affected by oligodontia, as well as different phenotypical manifestations, both systemic and oral. The previously undescribed PAX9 mutation was observed in the paired box (exon 2); this was a heterozygote transition of C175 to T, implying the change of arginine 59 for a termination codon. These results strongly suggested that the identified mutation was the etiological cause of the oligodontia. However, in two family members affected by both hypodontia and peg-shaped upper lateral incisors, no mutations in the PAX9 and MSX1 genes were identified. This fact underscores the importance that other presently unknown genes and developmental factors have in tooth development and in the etiology of dental anomalies.     

A novel nonsense variant in REEP6 is involved in a sporadic rod‐cone dystrophy case

Rod‐cone dystrophy (RCD), also called retinitis pigmentosa, is the most common form of progressive inherited retinal disorders secondary to photoreceptor degeneration. It is a genetically heterogeneous disease characterized by night blindness, followed by visual field constriction and, in most severe cases, total blindness. The aim of our study was to identify the underlying gene defect leading to severe RCD in a 60‐year‐old woman. The patient's DNA was investigated by targeted next generation sequencing followed by whole exome sequencing. A novel nonsense variant, c.267G>A p.(Trp89*), was identified at a homozygous state in the proband in REEP6 gene, recently reported mutated in 7 unrelated families with RCD. Further functional studies will help to understand the physiopathology associated with REEP6 mutations that may be linked to a protein trafficking defect.     

A recurrent frameshift mutation in exon 19 of the type VII collagen gene (COL7A1) in Mexican patients with recessive dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). In this study, we determined the molecular basis of autosomal recessive DEB in a 19-year-old Hispanic Mexican woman by PCR amplification of genomic DNA, heteroduplex analysis, and automated sequencing of heteroduplex bandshifts. This approach revealed a homozygous frameshift mutation, 2470insG, in exon 19 of COL7A1 and resulted in attenuated basement membrane zone expression of type VII collagen, a reduced number of anchoring fibrils at the dermal-epidermal junction, and a sub-lamina densa level of blister formation. Clinically, the patient had widespread trauma-induced skin fragility and complete loss of the nails, but had less pseudosyndactyly of the fingers and toes and milder mucosal involvement compared to most patients with the generalized form of this genodermatosis. We also screened 7 other Hispanic-Mexican patients with recessive DEB, none of whom were known to be related to this individual, for the mutation 2470insG using heteroduplex analysis and direct sequencing and detected this mutation on 7/14 alleles. Haplotype analysis using intragenic COL7A1 and flanking polymorphisms and microsatellite markers revealed that all the mutant alleles had arisen on similar allelic backgrounds, consistent with propagation of a common Hispanic Mexican ancestral haplotype. In view of the high allelic frequency of the mutation 2470insG in the patients studied, we recommend initial screening for this mutation when attempting to identify the molecular pathology of recessive DEB in Hispanic Mexican patients.     

Antithrombin gene Arg197Stop mutation-associated venous sinus thrombosis in a Chinese family

This study sought to elucidate the genetic correlation of cerebral venous sinus thrombosis caused by a hereditary antithrombin deficiency in a Chinese family,at the genetic and protein levels.A nonsense mutation from C to T on locus 6431 in exon 3B of the antithrombin gene was observed,leading to an arginine (CGA) to stop codon (TGA) change in the protein.This is the first report of this mutation in China.Ineffective heparin therapy in the propositus patient is associated with a lack of heparin binding sites after antithrombin gene mutation.Characteristic low intracranial pressure in the acute phase might be specific to this patient with cerebral venous sinus thrombosis.     

The study of the effect of splicing mutations in von Willebrand factor using RNA isolated from patients’ platelets and leukocytes

Summary. Background: In von Willebrand factor (VWF) the effect of mutations potentially affecting mRNA processing or splicing is less predictable than that of other mutations (e.g. nonsense or missense substitutions). Bioinformatic tools can provide a valuable means to determine the consequences of potential splice site mutations (PSSM), but functional studies are mandatory to elucidate the true effect of the variation detected. Objectives, patients and methods: After identification of PSSM in VWD patients, we began a systematic study of their in vivo effect in RNA extracted from the patients’ platelets and leukocytes. Results and conclusions: Thirteen pairs of primers were designed for full amplification of VWF mRNA by RT‐PCR that, after sequencing of aberrant products, enabled elucidation of the PSSM consequences for mRNA processing. This procedure was used to study seven different PSSM identified in four patients demonstrating diverse molecular mechanisms such as exon skipping (c.533?2A>G and c.8155+3G>C) and the activation of a cryptic splice site (c.7730?1G>C). No visible effect was evident for c.1533+15G>A and c.5170+10C>T and the consequence of c.[546G>A;7082?2A>G] was hidden by nonsense‐mediated mRNA decay (NMD). Results were compared with in silico predictions of four splice‐site analysis tools. We demonstrate selective degradation of VWF mRNA bearing PSSM by NMD for several mutations, which suggests that NMD represents a general mechanism for truncating mutations in VWF. Furthermore, because NMD efficiency varies between cell types, use of RNA from both platelets and leukocytes for in vivo study of VWF PSSM offers complementary results, particularly in cases in which NMD occurs in the allele carrying the mutation.     

Molecular modeling predicts structural changes in the A subunit of factor XIII caused by two novel mutations identified in a neonate with severe congenital factor XIII deficiency

Introduction

Coagulation factor XIII (FXIII) is a fibrin-stabilizing factor, which contributes to hemostasis, wound healing, and maintenance of pregnancy. Accordingly, patients with congenital FXIII deficiency manifest a life-long bleeding tendency, abnormal wound healing and recurrent miscarriage. In order to understand the molecular mechanisms of congenital FXIII deficiency, genetic analysis and molecular modeling were carried out in a Japanese male neonate with severe FXIII deficiency.

Methods and Results

Two novel mutations, Y204Stop (or Y204X, TAT to TAA) and S708R (AGC to AGG), were heterozygously identified by nucleotide sequencing analysis in exons V and XV of the gene for the A subunit of FXIII (FXIII-A). Y204X and S708R would lead to nonsense mediated mRNA decay and misfolding of the FXIII-A molecule, respectively. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, the presence of these mutations was confirmed both together in the proband and one each separately in either the maternal or paternal sides of his family. In addition, moderately decreased FXIII activity was associated with the presence of either mutation. Molecular modeling predicted that the mutant molecule of S708R would be structurally compromised by the substitution of the Ser with the larger extended bulky and positively charged Arg side-chain.

Conclusion

It is probable that the impaired tertiary structure of the mutant S708R molecule leads to its instability, which is at least in part responsible for the FXIII deficiency of this patient. This is consistent with the fact that the mutations and the reduced FXIII activities co-segregate among the patient's family members.     

Reducing the Toxicity of Designer Aminoglycosides as Nonsense Mutation Readthrough Agents for Therapeutic Targets

A significant proportion of genetic disease cases arise from truncation of proteins caused by premature termination codons. In eukaryotic cells some aminoglycosides cause readthrough of premature termination codons during protein translation. Inducing readthrough of these codons can potentially be of therapeutic value in the treatment of numerous genetic diseases. A significant drawback to the repeated use of aminoglycosides as treatments is the lack of balance between their readthrough efficacy and toxicity. The synthesis and biological testing of designer aminoglycoside compounds is documented herein. We disclose the implementation of a strategy to reduce cellular toxicity and maintain readthrough activity of a library of compounds by modification of the overall cationic charge of the aminoglycoside scaffold through ring I modifications.