A 10376 bp deletion of FECH gene responsible for erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP, MIM 177000) is an autosomal dominant disease with incomplete penetrance since the phenotypic expression requires coinheritance of a null allele and a wild-type low expressed allele of Ferrochelatase gene (FECH). In this study, we identify a peculiar mutation in a young Canadian patient of Italian origin. The patient had clinical and biochemical symptoms of EPP, the wild-type low expressed allele but at preliminary analysis no mutation in the promoter, in the entire coding region and in the splice junctions of the gene. Family studies of seven most common polymorphisms along the gene established absence of Mendelian segregation for the promoter polymorphism only. The intron 1 polymorphism appeared in heterozygosis suggesting an hypothetical deletion in the first region of the gene. In order to identify the size of this deletion, single nucleotide polymorphisms (SNPs) analysis was extended to the upstream N-asparaginyl-tRNA synthetase gene (NARS). We analyzed two polymorphisms in the last exon of this gene and a dizigous region was found in the patient. A Long-PCR with primers located in previously fixed heterozygous regions showed a 10,376 bp deletion (c.1-7887_67+2422del) that included a portion of the upstream intergenic region, the promoter, the exon 1 and a portion of intron 1. RNA analysis demonstrated that the lack of the entire promoter prevents the expression of the mutated allele, in fact the expression of the Ferrochelatase gene was decreased by half in the subjects carrying only the mutation compared to control.     

Association of a single-nucleotide polymorphism of the deleted-in-azoospermia-like gene with susceptibility to spermatogenic failure

Single-strand conformation polymorphism analysis of exon-containing genomic DNA segments of the deleted-in-azoospermia-like (DAZL) gene was performed in 160 infertile Taiwanese men presenting with severe oligozoospermia and nonobstructive azoospermia. An A-->G transition at nucleotide 386 in exon 3 was identified. The mutation is located within the RNA-recognition motif (aa 32-117) domain of the DAZL protein and will lead to Thr54-->Ala change (T54A) of DAZL protein. Analysis of cDNA from testicular tissue of infertile carriers showed absence of expression for the T54A allele, implying that the allele carrying T54A polymorphism is hardly, if ever, expressed. The frequencies of T54A allele in patients and the control group were 7.39% and 0.86%, respectively (P = 0.0003). The phenotypes varied significantly in cases with heterozygous T54A polymorphism, ranging from hypospermatogenesis and maturation arrest to Sertoli cell-only syndrome. A combination of DAZ gene deletion and T54A polymorphism did not worsen the phenotype. Our findings provide strong evidence for the role of the autosomal DAZL gene in human spermatogenesis.     

Hereditary spherocytosis with band 3 deficiency. Association with a nonsense mutation of the band 3 gene (allele Lyon), and aggravation by a low-expression allele occurring in trans (allele Genas)

We describe an 18-year-old with moderate hereditary spherocytosis. The condition was associated with a 35% decrease in band 3. The underlying mutation was Arg to stop at codon 150 (CGA-->TGA) and was designated R150X, which defined allele Lyon of the EPB3 gene. The inheritance pattern was dominant. However, the mother, who also carried the allele Lyon, had a milder clinical presentation and only a 16% decrease of band 3. We suggested that the father had transmitted a modifying mutation that remained silent in the heterozygous state. Nucleotide sequencing after single strand conformation polymorphism analysis of the band 3 cDNA and promoter region revealed a G-->A substitution at position 89 from the cap site in the 5'-untranslated region, designated 89G-->A, which defined allele Genas. A ribonuclease protection assay showed that (1) the allele Genas (father) resulted in a 33% decrease in the amount of band 3 mRNA, (2) the reduction caused by the allele Lyon (mother) was 42%, and (3) the compound heterozygous state for both alleles (proband) resulted in a 58% decrease. These results suggest that some mildly deleterious alleles of the EPB3 gene are compensated for by the normal allele in the heterozygous state. They are shown through the aggravation of the clinical picture, based on more obvious molecular alterations when they occur in trans to an allele causing a manifest reduction of band 3 membrane protein concentration.     

Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: identification and transfusion safety

The molecular backgrounds of variants encountered in Afro-Caribbean black individuals and associated with the production of clinically significant antibodies against high-incidence antigens (anti-RH18, anti-RH34) and against Rhe epitopes were determined. We showed that RH:-18 phenotypes are produced by 3 distinct RHCE alleles: ceEK carrying 48G>C (exon 1), 712A>G, 787A>G, 800T>A (exon 5); ceBI carrying 48G>C (exon 1), 712A>G (exon 5), 818C>T (exon 6), 1132C>G (exon 8); and the already known ceAR allele carrying 48G>C (exon 1), 712A>G, 733C>G, 787A>G, 800T>A (exon 5), and 916A>G (exon 6). The RH:-34 phenotype is produced by the (C)ce(s) haplotype described previously and composed of a hybrid D-CE(3-8)-D gene with 4 extra mutations next to a ce(s) allele (733C>G; exon 5) with an extra mutation in exon 7 (1006G>T). Partial Rhe with risk of immunization against lacking epitopes can be produced by the new ce(s) allele carrying an extra mutation in exon 3 (340C>T) and by the ceMO allele described previously. A population of sickle cell disease patients was screened to estimate the incidence of these rare alleles, with the conclusion that a procedure is required to detect the associated phenotypes in black donors to ensure transfusion safety for patients. We also described a new variant [ce(s)(748)] and variants carrying different altered alleles in nonimmunized patients and for whom the risk of immunization is discussed.     

Novel mutations in the Factor VII gene of Taiwanese Factor VII-deficient patients

The genetic defects of four Taiwanese patients with factor VII (FVII) deficiency were studied. FVII activity and antigen levels were < 1 u/dl and 125.7 u/dl (patient I), < 1 u/dl and < 1 u/dl (patient II), 3.4 u/dl and 5.9 u/dl (patient III), and 1.2 u/dl and 30.4 u/dl (patient IV) respectively. The 5' flanking region, and all exons and junctions were amplified using polymerase chain reaction and sequenced. Patient I was homozygous for a 10824C-->A transversion with Pro303-->Thr mutation in exon 8. In patient II, a heterozygous transversion, 9007+1G-->T at the IVS6, a heterozygous decanucleotide insertion polymorphism at -323 (both mutations present in his father) and a heterozygous deletion, del TC (26-27) in exon 1A (originating from his mother) were identified. Patient III had a homozygous 10961T-->G transversion with His348-->Gln mutation in exon 8. Patient IV had a heterozygous 10902T-->G transversion with Cys329-->Gly mutation in exon 8 (transmitted to her second son) and a heterozygous decanucleotide insertion polymorphism at -323 (transmitted to her third son). All but one of the FVII gene mutations detected in the four patients have not been previously reported. In conclusion, four novel mutations of the FVII gene in Taiwanese, including two missense mutations in exon 8, one point mutation at the exon 6 splice site and one deletion in exon 1A, were identified.     

Severe obesity and insulin resistance due to deletion of the maternal Gsalpha allele is reversed by paternal deletion of the Gsalpha imprint control region

The G protein alpha-subunit G(s)alpha mediates receptor-stimulated cAMP production and is imprinted with reduced expression from the paternal allele in specific tissues. Disruption of the G(s)alpha maternal (but not paternal) allele leads to severe obesity, hypertriglyceridemia, and insulin resistance in mice and obesity in patients with Albright hereditary osteodystrophy. Paternal deletion of a G(s)alpha imprint control region (1A) leads to loss of tissue-specific G(s)alpha imprinting. To determine whether the metabolic abnormalities resulting from disruption of the G(s)alpha maternal allele could be reversed by loss of paternal G(s)alpha imprinting, females with a heterozygous G(s)alpha exon 1 deletion were mated to males with heterozygous deletion of the imprint control region (1A) to generate mice with maternal G(s)alpha deletion (E1(m-)), paternal 1A deletion (1A(p-)), double mutants (E1(m-):1A(p-)), and wild type. E1(m-) mice developed obesity, glucose intolerance, insulin resistance, and hypertriglyceridemia, which were all normalized by the paternal 1A deletion in E1(m-):1A(p-) mice. Obesity in E1(m-) was associated with reduced energy expenditure and sympathetic nerve activity, and these were also normalized in E1(m-):1A(p-) mice. 1A(p-) mice had reduced body weight associated with proportional decreases in fat and lean mass as well as increased activity levels. The metabolic phenotype resulting from maternal G(s)alpha deletion is rescued by a genetic lesion that leads to loss of tissue-specific G(s)alpha imprinting, consistent with this phenotype being a direct consequence of G(s)alpha imprinting in one or more specific tissues.     

An alternative fast and convenient genotyping method for the screening of angiotensin converting enzyme gene polymorphisms.

Insertion/deletion (I/D) polymorphisms in intron 16 of the angiotensin converting enzyme gene (ACE) are associated with the plasma angiotensin converting enzyme (ACE) levels, and individuals with the DD allele have been reported to be more susceptible to cardiovascular disease than those without. The conventional genotyping method for the screening of I/D polymorphisms, which involves polymerase chain reaction (PCR)-gel electrophoresis, is laborious and time-consuming. In this study, we assessed the use of TaqMan-PCR genotyping for the screening of I/D polymorphisms as a replacement for the conventional method. We genotyped seven single nucleotide polymorphisms (SNPs) in linkage disequilibrium (LD) with the I/D polymorphisms, and calculated the LD coefficients of the I/D polymorphisms. We found that three polymorphisms, rs4331, rs4334 and rs4341, exhibited the highest LD coefficients (D' = 1.000; r2 = 0.967) and that the genotyping of rs4341 by the TaqMan-PCR method yielded the best discrimination among the different genotypes. Genotyping of 511 samples took only 2 h and the amount of DNA required for each test was only 6 ng by the TaqMan-PCR method using rs4341. In the course of this study, we identified a novel additional polymorphism (a deletion of six amino acids) in exon 13, near rs4316. The deletion allele encoded the testicular ACE, but not the plasma ACE. We concluded that genotyping of the rs4341 ACE polymorphism by the TaqMan-PCR method is a fast and convenient alternative method for direct I/D genotyping. We also concluded that testicular ACE may manifest a deletion of six amino acids that may result in deleterious function of this enzyme.     

A silenced allele in the Colton blood group system

Background The antigens of the Colton blood group system, Co^a and Co^b, are encoded by a single gene that produces the aquaporin‐1 (AQP1) protein, a water channel‐forming protein, and are characterized by a single nucleotide polymorphism (SNP). A healthy Caucasoid blood donor originally typed as Co(a?b?) with commercial anti‐Co^b typed Co(a?b+) when retested with another anti‐Co^b. Retyped with two different molecular biology methods, the sample came out Co^a/Co^b. With the aim of understanding these discrepancies, serological, cytometric and molecular biology tests were carried out. Methods Absorption/elution studies with propositus red cells and controls were performed. The region spanning exon 1 to exon 4 of the Colton gene was sequenced, and flow cytometry analyses were carried out. Results Absorption/elution studies showed the absence of Co^a and a weak expression of Co^b. DNA sequencing confirmed a CT heterozygosity at nucleotide position 134 (i.e. Co^a/Co^b), and an additional heterozygous CT was found at position 112. The presence of the Co^b allele that encodes for the Co^b antigen was confirmed. The new allele has the base cytosine at nucleotide 134 (Co^a), in cis with the new nucleotide 112T. The nucleotide substitution 112C>T causes a missense mutation leading to an amino acid change from proline (CCG) to serine (TCG) at codon 38. Conclusion The substitution found at codon 38 results in a modified AQP1 protein which explains the Co(a?b+) phenotype and possibly the weak expression of Co^b.     

The common C49620T polymorphism in the sulfonylurea receptor gene (ABCC8), pancreatic beta cell function and long-term diabetic complications in obese patients with long-lasting type 2 diabetes mellitus.

HYPOTHESIS: A gene polymorphism associated with accelerated beta-cell failure may lead to a more rapid development of long-term complications of type 2 diabetes (T2DM) due to a worse metabolic control of the disease. AIM OF THE STUDY: Evaluation of an association between the intronic C49620T (exon 16 -3c-->t) polymorphism in the ABCC8 (SUR1) gene and beta-cell function, as well as the prevalence of long-term diabetic complications in obese patients with long-lasting type 2 diabetes. METHODS: Two hundred and fifteen obese patients with at least a 10-year history of T2DM were thoroughly characterized clinically. In all the patients the intravenous glucagon test was performed and the C49620T ABCC8 polymorphism was assessed. Subgroups of patients, classified either according to genotype or to allele carriage, were compared. RESULTS: No difference was found between the groups in variables describing beta-cell function and the prevalence of chronic diabetic complications, with the exception of a significantly lower incidence of brain stroke in CC homozygotes than in patients carrying T allele (CT+TT). Body mass index was higher in patients carrying C allele than in TT homozygotes. HDL-cholesterol was higher in CT heterozygous than in homozygous CC or TT patients. CONCLUSIONS: There is no association between the ABCC8 polymorphism gene and the beta-cell function or the prevalence of chronic diabetic complications in obese patients with long-term T2DM, except for brain stroke. The results might suggest that the homozygous CC subjects are at lower risk of the complication, but additional studies are warranted to test this finding.     

A complex heterozygous mutation of His373Leu and Asp487-Ser488-Phe489 deletion in human cytochrome P450c17 causes 17alpha-hydroxylase/17,20-lyase deficiency in three Chinese sisters

Cytochrome P450c17 deficiency is one of the rare forms of enzyme disorders in steroid biosynthesis, resulting from defects in 17alpha-hydroxylase and 17,20-lyase activities. The disease is caused by the mutations in CYP17 gene, inherited in an autosomal recessive pattern. We reported a Chinese family with three sisters suffering from P450c17 deficiency based on their clinical features and molecular genetics. The patients were found to be compound heterozygotes with two different mutations. Screened by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), a heterozygous point mutation His373Leu was detected in the exon 6 of CYP17 gene which was proved to be derived from paternal allele. The other allele contained nine-base pair deletion, located in exon 8, eliminating codons 487-489 (Asp-Ser-Phe) near the carboxy-terminus of P450c17. The mother and the brother have been demonstrated to be carriers of deletion mutation through restriction enzyme analysis. Both mutations have been reported previously in Asia. This is the first report of the molecular genetic study of 17alpha-hydroxylase/17,20-lyase deficiency in mainland China with a novel compound heterozygous mutation.     

Tumor necrosis factor receptor II gene polymorphism and severity of rheumatoid arthritis

OBJECTIVE: The gene encoding tumor necrosis factor receptor type II (TNFRII) is a strong candidate in the pathogenesis of rheumatoid arthritis (RA). An association between a single-nucleotide polymorphism (196M/R) in exon 6 of the TNFRII gene and familial RA was recently reported. The present study was undertaken to test the hypothesis that there is an association between this polymorphism and the severity of RA. METHODS: One hundred two white patients with early RA were included in this prospective study. The French version of the Health Assessment Questionnaire (F-HAQ) and a radiographic damage score (modified Sharp/van der Heijde method) were used to quantify the functional and structural severity of RA at baseline and after 4 years of followup. TNFRII 196M/R polymorphism genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: Among the 102 patients with RA, 63 (61.8%) were homozygous for the 196M allele, 36 (35.3%) were heterozygous for alleles 196M and 196R, and 3 (2.9%) were homozygous for the 196R allele. At baseline, the median radiographic and F-HAQ scores did not differ between RA patients who carried the 196R allele and those who did not. After 4 years of followup, the F-HAQ score was higher in RA patients carrying the 196R allele (median 1 [interquartile range (IQR) 0.125, 1.375]) than in noncarriers (0.375 [IQR 0, 1]) (P = 0.02), while the median radiographic score did not differ between RA patients who carried the 196R allele and those who did not. CONCLUSION: The results of the present study support the hypothesis that there is an association between the TNFRII 196 M/R gene polymorphism and the functional severity of early RA.     

Scianna antigens including Rd are expressed by ERMAP

The Scianna blood group encompasses the high-frequency antigens Sc1 and Sc3 and the low-frequency antigen Sc2. Another low-frequency antigen Rd (Radin) was suggested to belong to the Scianna blood group. The molecular basis of the Scianna blood group was unknown. The erythrocyte membrane-associated protein (ERMAP) shared the genomic location, protein product size, and localization to the red blood cell (RBC) membrane surface with Scianna. The ERMAP gene was sequenced in probands with known Scianna and Radin phenotypes. In a Sc:-1,-2 proband, only an ERMAP allele with a 2-bp deletion in exon 3 causing a frameshift could be detected. A Sc:-1,2 proband was homozygous for the ERMAP(Gly57Arg) allele. An Rd(+) proband was heterozygous for the ERMAP(Pro60Ala) allele. Polymerase chain reaction with sequence-specific priming (PCR-SSP) systems was developed to detect the Sc2 and Rd alleles of the ERMAP gene. The 2 alleles occurred with about 1% and less than 1% frequency in the population, which was compatible with the frequency of the Sc2 and Rd antigens known in whites. Two Sc2(+) and one Rd(+) samples that were found by genotyping were confirmed by serology. The antigens of the Scianna blood group include Rd and are expressed by the human ERMAP protein. Sc2 is caused by an ERMAP(Gly57Arg) allele and Rd by an ERMAP(Pro60Ala) allele. Scianna is the last of the previously characterized protein-based blood group systems whose molecular basis was discerned. Hence, the phenotype prediction by genotyping became possible for all human blood group systems encoded by proteins.     

SNRPN基因在HepG2细胞中的印迹状态研究

目的:研究小核核糖核蛋白多肽N基因(SNRPN)在肝癌肿瘤HepG2细胞株的表达及基因印迹状态.方法:采用RT-PCR方法检测出SNRPN基因在肝癌肿瘤HepG2细胞株中的表达状况,对HepG2细胞株基因组DNA和cDNA中的SNRPN基因外显子4nt1654312位点用RT-PCR为基础的RFLP方法进行基因分型.结果:HepG2细胞稳定表达SNRPN,SNRPN外显子4nt1654312(数字依据NT_026446,SNPrs705)为杂合子(C/T);RT-PCR为基础的RFLP分析表明,SNRPN的双等位基因中只有T等位基因产生mRNA转录本.结论:SNRPN基因在HepG2肝癌细胞株中有表达,其基因印迹状态未丢失.     

Reduced expression of a human V beta 6.1 T-cell receptor allele.

We have previously described an allelic polymorphism in the V beta 6.1 T-cell receptor gene. The V beta 6.1B allele is associated with disease in a subgroup of patients with juvenile rheumatoid arthritis. Limited sequence data demonstrated nucleotide differences that resulted in two amino acid changes between the two alleles in positions predicted to be important in major histocompatibility complex/antigen recognition. The present study demonstrates substantially reduced expression of mRNA from the disease-associated allele (V beta 6.1B) in peripheral blood and thymic tissue. The complete genomic sequence of both alleles revealed two additional amino acid changes in the V beta 6.1B gene as well as nucleotide differences in the promoter and intron. A cysteine-to-arginine substitution at position 92 in the disease-associated allele makes this a non-functional beta chain, since this conserved cysteine is involved with disulfide bonding to cysteine-23 to form an immunoglobulin-like domain structure, thus resulting in a potential hole in the T-cell receptor repertoire.     

Ethnic distribution of allele aLELY, a low-expression allele of red-cell spectrin a-gene

Summary. Alleleα^LELY low-expression allele of erythroid spectrin a-chain. It carries mutations both in exon 40 and intron 45 and is associated with partial skipping of exon 46. Allele α^LELY remains asymptomatic by itself. In contrast, it enhances the expression level of deleterious α-alleles occurring in trans , and as such has clinical importance. The aim of this study was to evaluate the incidence of allele _QLBLY _mvarj_ous ethnic groups, i.e. Caucasians, African Blacks, Japanese and Chinese. Allele Q^LELY occurred in all groups investigated with a fairly uniform frequency: 31%, 21%, 20% and 22%, respectively. Mutations in exon 40 and intron 45 appeared linked to one another without exception. Partial skipping of exon 46 or the low-expression feature, whenever they could be assessed, were invariably observed. Allele α^LELY appears to be an ancient and stable allele.