Large deletion of androsterone UDP-glucuronosyltransferase gene in the inherited deficient strain of Wistar rats.

LA Wistar rats have a deficiency of androsterone UDP-glucuronosyltransferase (UDPGT) and are present in Wistar rat colonies around the world. In order to clarify the molecular mechanism of the deficiency, androsterone UDPGT cDNA clone, pGT2 was isolated from rat liver cDNA library and was digested with restriction enzymes to afford three probes for Northern and Southern blot analyses in HA (normal), heterozygous LA and LA Wistar rats. In Northern blot analysis, androsterone UDPGT mRNA was totally absent in LA Wistar rat liver. Southern blot analysis suggested a large deletion of androsterone UDPGT gene in the rats. Genomic DNA amplifications with synthetic primers which have nucleotide sequences corresponding to the 5'-region of androsterone UDPGT cDNA, suggested that androsterone UDPGT gene has exon 1 with a length of some 700 bp and that this exon is deleted in LA Wistar rats. Based on these lines of evidence, it is concluded that the large portion of androsterone UDPGT gene is deleted in LA Wistar rats, which results in the absence of androsterone UDPGT mRNA and consequently the corresponding enzyme protein.     

Detection of somatic and germline mosaicism for the LAMP2 gene mutation c.808dupG in a Chinese family with Danon disease

Danon disease is a rare X-linked lysosomal storage disease characterized by hypertrophic cardiomyopathy, myopathy and mental retardation, and is due to a primary defect in lysosome-associated membrane protein-2 (LAMP 2). More than 26 mutations in the LAMP2 gene have been described, including a small number of de novo mutations, some of which are suspected to be caused by germline mosaicism. Here, we describe the first molecularly documented evidence of somatic mosaicism for a LAMP2 mutation, identified in the asymptomatic mother of a boy with Danon disease caused by the frameshift mutation c.808dupG (p.A270Gfx3) within exon 6. In addition, in order to gain insight into the possible explanation for the mother's lack of phenotype, the level of somatic mosaicism and the X-chromosome inactivation pattern were investigated. This study provides new insight into the causes of phenotypic variability in female mutation-carriers and underlines the importance of parental molecular testing for accurate genetic counseling for Danon disease.     

Restoration of complex V deficiency caused by a novel deletion in the human TMEM70 gene normalizes mitochondrial morphology

We report a fragmented mitochondrial network and swollen and irregularly shaped mitochondria with partial to complete loss of the cristae in fibroblasts of a patient with a novel TMEM70 gene deletion, which could be completely restored by complementation of the TMEM70 genetic defect. Comparative genomics analysis predicted the topology of TMEM70 in the inner mitochondrial membrane, which could be confirmed by immunogold labeling experiments, and showed that the TMEM70 gene is not restricted to higher multi-cellular eukaryotes. This study demonstrates that the role of complex V in mitochondrial cristae morphology applies to human mitochondrial disease pathology.     

A large deletion/insertion-induced frameshift mutation of the androgen receptor gene in a family with a familial complete androgen insensitivity syndrome

Androgen insensitivity syndrome (AIS) is an X-linked recessive genetic disorder with a normal 46, XY karyotype caused by abnormality of the androgen receptor (AR) gene. One Chinese family consisting of the proband and 5 other members with complete androgen insensitivity syndrome (CAIS) was investigated. Mutation analysis by DNA sequencing on all 8 exons and flanking intron regions of the AR gene revealed a unique large deletion/insertion mutation in the family. A 287 bp deletion and 77 bp insertion (c.933_1219delins77) mutation at codon 312 resulted in a frameshift which caused a premature stop (p.Phe312Aspfs*7) of polypeptide formation. The proband's mother and grandmother were heterozygous for the mutant allele. The proband's father, uncle and grandfather have the normal allele. From the pedigree constructed from mutational analysis of the family, it is revealed that the probably pathogenic mutation comes from the maternal side.     

Identification of a deletion in the NDUFS4 gene using array-comparative genomic hybridization in a patient with suspected mitochondrial respiratory disease

We evaluated a patient, born after a normal 38-week pregnancy, with psychomotor retardation, poor coordination of ocular movements, recurrent vomiting and severe lactic acidosis. The patient was admitted to hospital at 2 months of age because of a mitochondrial-like syndrome and died at the age of 4.5 months. Array-comparative genomic hybridization (a-CGH) analysis revealed a homozygous deletion in 5q11.2 involving NADH dehydrogenase (ubiquinone) Fe–S protein 4, 18 kDa (NADH-coenzyme Q reductase; NDUFS4). Both parents were heterozygous for the mutation. The array revealed a deletion of ~ 32 kb that includes exon 2 of NDUFS4 subsequently confirmed by real time-PCR and multiplex PCR. NDUFS4 was previously correlated to Leigh syndrome since mutations in this gene block the assembly of complex I. This result demonstrates the relevance of a-CGH screening in patients affected by metabolic disorders of unknown etiology.     

A submicroscopic deletion involving part of the CREBBP gene detected by array-CGH in a patient with Rubinstein-Taybi syndrome

We report a girl with Rubinstein-Taybi syndrome (RSTS) who was found to have copy number loss on 16p13.3 by array-CGH. She has developmental delay and other features of RSTS including downslanting palpebral fissures, a prominent nose with the nasal septum extending below the alae nasi, broad thumbs and big toes, postaxial polydactyly of the right foot and constipation from birth. We report the junction sequence across the breakpoint region for a microdeletion in RSTS. The sequencing results also showed that the deletion was 81.4kb involving three genes DNASE 1, TRAP 1, and CREBBP.     

Polymorphisms of the insulin-like growth factor-binding protein 3 gene (IGFBP3) in gayal (Bos frontalis)

The gene coding for insulin-like growth factor-binding protein 3 (IGFBP3) is important for regulation of growth, development and metabolism in mammals. The present investigation was conducted to study nucleotide polymorphism of the IGFBP3 in gayal (Bos frontalis) and to compare the variations with those which occur in other ruminants. A fragment of 645 base pairs of the IGFBP3 covering a part of exon 2, the complete intron 2 and exon 3 and a part of intron 3 was amplified, sequenced (n=46) and digested (n=79) with HaeIII restriction enzyme from 125 collected gayal samples. Nine single nucleotide polymorphisms (SNPs) [C14T, A122C, C137T, G144C, C155T, G213A, C279A, G334A and G460A] were identified and located in intron 2, revealing high genetic variability. The alignment of nucleotide sequences was found to be very similar to those for other bovid species. Sequencing and HaeIII digestion showed that frequency of alleles C and A [consisting of fragments of sizes 56, 64, 228, 264, 282, 298 and 497 bp (CC genotype)] was 0.96 and 0.04 for the SNP C279A. Moreover, the genotype frequency of the SNP C279A in gayal was compared with that in other ruminants and it appears that this polymorphism may be associated with low fat content and rapid growth in this rare species.     

Characterization of seven novel mutations on the HEXB gene in French Sandhoff patients

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by mutations in the HEXB gene encoding the beta subunit of hexosaminidases A and B, two enzymes involved in GM2 ganglioside degradation. Eleven French Sandhoff patients with infantile or juvenile forms of the disease were completely characterized using sequencing of the HEXB gene. A specific procedure was developed to facilitate the detection of the common 5′-end 16 kb deletion which was frequent (36% of the alleles) in our study. Eleven other disease-causing mutations were found, among which four have previously been reported (c.850C>T, c.793T>G, c.115del and c.800_817del). Seven mutations were completely novel and were analyzed using molecular modelling. Two deletions (c.176del and c.1058_1060del), a duplication (c.1485_1487dup) and a nonsense mutation (c.552T>G) were predicted to strongly alter the enzyme spatial organization. The splice mutation c.558+5G>A affecting the intron 4 consensus splice site led to a skipping of exon 4 and to a truncated protein (p.191X). Two missense mutations were found among the patients studied. The c.448A>C mutation was probably a severe mutation as it was present in association with the known c.793T>G in an infantile form of Sandhoff disease and as it significantly modified the N-terminal domain structure of the protein. The c.171G>C mutation resulting in a p.W57C amino acid substitution in the N-terminal region is probably less drastic than the other abnormalities as it was present in a juvenile patient in association with the c.176del. Finally, this study reports a rapid detection of the Sandhoff disease-causing alleles facilitating genetic counselling and prenatal diagnosis in at-risk families.     

The Jumonji gene family in Crassostrea gigas suggests evolutionary conservation of Jmj-C histone demethylases orthologues in the oyster gametogenesis and development

Jumonji (Jmj) proteins are histone demethylases, which control the identity of stem cells. Jmj genes were characterized from plants to mammals where they have been implicated in the epigenetic regulation of development. Despite the Pacific oyster Crassostrea gigas representing one of the most important aquaculture resources worldwide, the molecular mechanisms governing the embryogenesis and reproduction of this lophotrochozoan species remain poorly understood. However, annotations in the C. gigas EST library suggested the presence of putative Jumonji genes, raising the question of the conservation of this family of histone demethylases in the oyster.     

Effect of interleukin-17A and interleukin-17F gene polymorphisms on the risk of gastric cancer in a Chinese population

We selected six tagged single-nucleotide polymorphisms (SNPs) in the IL-17A and IL-17F genes, and evaluated the relationship between the six common SNPs and H. pylori infection, tobacco smoking and subsites of gastric cancer in gastric cancer patients. Genotyping of IL-17A (rs2275913, rs3748067 and rs3819025) and IL-17A (rs763780, rs9382084, and rs12203582) was performed in a 384-well plate format on the MassARRAY® platform. An unconditional multiple logistical regression model was performed to determine the association between IL-17A and IL-17F genetic variations and gastric cancer risk. Unconditional logistic regression analysis showed that subjects carrying the rs2275913AA and rs3748067 TT genotypes were 1.70 and 3.45 times more likely to develop gastric cancer. Furthermore, rs2275913 and rs3748067 genetic variants significantly interacted with H. pylori infection on the risk of gastric cancer. The interaction between rs3748067 and rs9382084 genetic variants and tobacco smoking trend was significant. In addition, rs2275913, rs3748067 and rs9382084 genetic variants were only associated with non-cardia gastric cancer. The findings suggest that the rs2275913, rs3748067 and rs9382084 polymorphisms increase the risk of gastric cancer, and they interact with H. pylori infection, tobacco smoking and subsites of gastric cancer. These findings could be helpful in identifying individuals at increased risk for developing gastric cancer.     

UGT1A1 sequence variants associated with risk of adult hyperbilirubinemia: A quantitative analysis

Backgrounds and Aims

UDP-glucuronosyltransferase 1 A1 (UGT1A1) is an enzyme that transforms small lipophilic molecules into water-soluble and excretable metabolites. UGT1A1 polymorphisms contribute to hyperbilirubinemia. This study quantitatively associated UGT1A1 variants in patients with hyperbilirubinemia and healthy subjects.

Methods

A total of 104 individuals with hyperbilirubinemia and 105 healthy controls were enrolled for genotyping and DNA sequencing UGT1A1 sequence variants, including the Phenobarbital Response enhancer module (PBREM) region, the promoter region (TATA box), and the 5 exons for quantitative association with hyperbilirubinemia.

Results

Eleven UGT1A1 variants were revealed in the case and control subjects, four of which were novel coding variants. A variant of PBREM (UGT1A1*60) was found in 47.6% of the patients, a TA repeat motif in the 5-primer promoter region [A(TA)₇TAA,UGT1A1*28] was found in 27.9% of the patients, and p.G71R (UGT1A1*6) was in 33.2% of the patients. For the healthy controls, the frequency of UGT1A1*60, UGT1A1*28 and UGT1A1*6 was 26.7%, 9.0% and 15.7%, respectively. Homozygous UGT1A1*28 and homozygous UGT1A1*6 were significantly associated with the risk of adult hyperbilirubinemia, with an odds ratio (OR) of 17.79 (95% CIs, 2.11–133.61) and 14.93 (95% CIs, 1.83–121.88), respectively. Quantitative analysis showed that sense mutation (including UGT1A1*6) and UGT1A1*28/*28, but not UGT1A1*60/*60 or UGT1A1*1/*28, was associated with increased serum total bilirubin (TB) levels. High linkage disequilibrium occurred between UGT1A1*60 and UGT1A1*28 (D′ = 0.964, r² = 0.345).

Conclusions

This study identified four novel UGT1A1 coding variants, some of which were associated with increased serum TB levels. A quantitative approach to evaluate adult hyperbilirubinemia provides a more vigorous framework for better understanding of adult hyperbilirubinemia genetics.     

Novel homozygous mutations in the WNT10B gene underlying autosomal recessive split hand/foot malformation in three consanguineous families

Split-hand/split-foot malformation (SHFM), representing variable degree of median clefts of hands and feet, is a genetically heterogeneous group of limb malformations with seven loci mapped on different human chromosomes. However, only 3 genes (TP63, WNT10B, DLX5) for the seven loci have been identified.     

Extension of the molecular analysis to the promoter region of the iduronate 2-sulfatase gene reveals genomic alterations in mucopolysaccharidosis type II patients with normal coding sequence

Hunter disease or mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by the deficit of the enzyme iduronate-2-sulfatase (IDS), involved in the catabolism of the glycosaminoglycans heparan and dermatan sulfate. Our aim was to search for molecular defects in the promoter region of the IDS gene in patients with previous biochemical diagnosis of MPS II and after we sequenced the whole IDS coding region and the exon/intron boundaries without detecting any pathogenic mutations. Screening of the promoter region of four patients detected in two of them a 178 bp deletion and in the other two a single nucleotide substitution 818 bp upstream of the coding region. The latter had never been described before in MPS II patients and it turned out to be a polymorphism. Our experience suggests that MPS II patients with no mutations detected in the IDS coding region should be screened in the promoter region of the gene. Findings will hopefully help to clarify the relationship between genotype and phenotype and will be useful for the correct molecular diagnosis of Hunter patients and the identification of female carriers, the latter particularly important for genetic counseling.     

The complete mitochondrial genome of the Chinese hook snout carp Opsariichthys bidens (Actinopterygii: Cypriniformes) and an alternative pattern of mitogenomic evolution in vertebrate

The complete mitochondrial genome sequence of the Chinese hook snout carp, Opsariichthys bidens, was newly determined using the long and accurate polymerase chain reaction method. The 16,611-nucleotide mitogenome contains 13 protein-coding genes, two rRNA genes (12S, 16S), 22 tRNA genes, and a noncoding control region. We use these data and homologous sequence data from multiple other ostariophysan fishes in a phylogenetic evaluation to test hypothesis pertaining to codon usage pattern of O. bidens mitochondrial protein genes as well as to re-examine the ostariophysan phylogeny. The mitochondrial genome of O. bidens reveals an alternative pattern of vertebrate mitochondrial evolution. For the mitochondrial protein genes of O. bidens, the most frequently used codon generally ends with either A or C, with C preferred over A for most fourfold degenerate codon families; the relative synonymous codon usage of G-ending codons is greatly elevated in all categories. The codon usage pattern of O. bidens mitochondrial protein genes is remarkably different from the general pattern found previously in the relatively closely related zebrafish and most other vertebrate mitochondria. Nucleotide bias at third codon positions is the main cause of codon bias in the mitochondrial protein genes of O. bidens, as it is biased particularly in favor of C over A. Bayesian analysis of 12 concatenated mitochondrial protein sequences for O. bidens and 46 other teleostean taxa supports the monophyly of Cypriniformes and Otophysi and results in a robust estimate of the otophysan phylogeny.     

Interaction between gene A-positive Helicobacter pylori and human leukocyte antigen II alleles increase the risk of Graves disease in Chinese Han population: An association study

Objective

The current study explored the correlation of Helicobacter pylori and the polymorphisms of human leukocyte antigen II (HLA-II) alleles with Graves disease (GD).

Methods

A total of 216 patients with GD were recruited. 102 healthy volunteers constituted the control group. Levels of H. pylori immunoglobulin G (IgG) antibodies and H. pylori cytotoxin-associated gene A (CagA) IgG antibodies were detected using enzyme-linked immunosorbent assays. Molecular typing of the HLA-II alleles was conducted using polymerase chain reaction with sequence specific primers.

Results

H. pylori, particularly CagA-positive strains, HLA-DQA1*0201, and HLA-DQA1*0501 were associated with GD (P = 0.015, OR = 1.811; P = 0.000, OR = 3.085; P = 0.000, OR = 0.315; and P = 0.004, OR = 2.844, respectively). Patients with CagA-positive H. pylori and negative HLA-DQA1*0201 or positive HLA-DQA1*0501 were more likely exposed to GD compared with those with only one of these indices.

Conclusion

CagA-positive H. pylori, negative HLA-DQA1*0201, or positive HLA-DQA1*0501 may increase the risk of GD.     

A novel heterozygous mutation of the AIRE gene in a patient with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED)

Background

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) is an autosomal recessive disease due to mutations of the autoimmune regulator (AIRE) gene. Typical manifestations include candidiasis, Addison's disease, and hypoparathyroidism. Type 1 diabetes, alopecia, vitiligo, ectodermal dystrophy, celiac disease and other intestinal dysfunctions, chronic atrophic gastritis, chronic active hepatitis, autoimmune thyroid disorders, pernicious anemia and premature ovarian failure are other rare associated diseases although other conditions have been associated with APECED.

Case presentation

What follows is the clinical, endocrinological and molecular data of a female APECED patient coming from Lithuania. The patient was affected by chronic mucocutaneous candidiasis, hypoparathyroidism and pre-clinical Addison's disease. Using direct sequencing of all the 14 exons of the AIRE gene in the patient's DNA, we identified in exon 6 the known mutation c.769 C>T (p.Arg257X) in compound heterozygosity with the newly discovered mutation c.1214delC (p.Pro405fs) in exon 10. The novel mutation results in a frameshift that is predicted to alter the sequence of the protein starting from amino acid 405 as well as to cause its premature truncation, therefore a non-functional Aire protein.

Conclusions

A novel mutation has been described in a patient with APECED with classical clinical components, found in compound heterozygosity with the c.769 C>T variation. Expanded epidemiological investigations based on AIRE gene sequencing are necessary to verify the relevancy of the novel mutation to APECED etiopathogenesis in the Lithuanian population and to prove its diagnostic efficacy in association with clinical and immunological findings.     

Plasmodium yoelii: the effect of second blood meal and anti-sporozoite antibodies on development and gene expression in the mosquito vector, Anopheles stephensi

The sporogonic development of the malaria parasite takes place in the mosquito and a wide range of factors modulates it. Among those, the contents of the blood meal can influence the parasite development directly or indirectly through the mosquito response to the infection. We have studied the effect of a second blood meal in previously infected mosquitoes and the effect of anti-sporozoite immune serum on parasite development and mosquito response to the infection. The prevalence and intensity of infection and gene expression of both Plasmodium yoelii and Anopheles stephensi was analyzed. We verified that a second blood meal and its immune status interfere with parasite development and with Plasmodium and mosquito gene expression.