Combined test for UGT1A1 -3279T-->G and A(TA)nTAA polymorphisms best predicts Gilbert's syndrome in Italian pediatric patients

Gilbert's syndrome is a common hereditary chronic or recurrent, mild unconjugated hyperbilirubinemia. Polymorphisms in the bilirubin uridine diphosphate glucuronosyl transferase gene (UGT1A1) causing a decreased enzyme activity are associated with susceptibility to the syndrome. Homozygosity for TA(7) allele of the A(TA)(n)TAA promoter polymorphism is found in the majority of Caucasian patients. We sought to investigate the role of three UGT1A1 polymorphisms (A[TA](n)TAA, -3279T-->G, and G71R) in the susceptibility to Gilbert's syndrome in 53 Italian pediatric subjects compared to 83 unaffected controls. Carriage of two TA(n) risk alleles (TA(7) and TA(8)) and -3279G homozygosity were similarly associated with hyperbilirubinemia (odds ratio [OR] = 11.59, 95% confidence interval [CI] = 4.80-27.98; p < 0.001, and OR = 11.51, 95% CI = 5.06-26.19; p < 0.001, respectively). Homozygosity for both TA7 and -3279G was associated with the highest relative risk estimate (OR = 19.23, 95% CI = 7.34-50.4; p < 0.001), but a significant association was found also for TA7 heterozygosity combined with -3279G/G genotype (OR = 7.98, 95% CI = 2.54-25.06; p < 0.001). The G71R variant was found only in two controls. Our results demonstrate that genotyping of both UGT1A1 A(TA)(n)TAA and -3279T-->G polymorphisms best defines genetic susceptibility to Gilbert's syndrome in Caucasian pediatric patients, and the TA7 heterozygous genotype combined with homozygosity for the -3279G allele can also be associated with pediatric mild hyperbilirubinemia.     

UDP-glucuronosyltransferase 1A1 (UGT1A1) gene haplotypes and their effect on serum bilirubin concentration in healthy Indian adults

The aim of the present study was to investigate the allele and genotype frequencies and haplotype structures of the variants in the UGT1A1 gene and their association with serum bilirubin levels in healthy adults. Total serum bilirubin levels were measured in 300 healthy adults (normal hematology and liver function test) and genotyping of seven SNPs was performed by PCR-RFLP, Gene Scan analysis and direct sequencing on the ABI Prism 310 Genetic Analyzer. Of the seven SNPs, four were found to be polymorphic and the frequencies of minor alleles were 0.336, 0.431, 0.353 and 0.066 for − 53(TA)7, − 3279G, − 3156A and 211A respectively. Individuals who carried the − 53(TA)7, − 3279G and − 3156A mutant alleles in homozygous or heterozygous states had significantly higher mean serum bilirubin levels. Five major promoter haplotypes were observed: − 53(TA)6/− 3279T/− 3156G was the most common haplotype, followed by − 53(TA)7/− 3279G/− 3156A, − 53(TA)6/− 3279G/− 3156G, − 53(TA)6/− 3279G/− 3156A and − 53(TA)7/− 3279T/− 3156G with an estimated frequency of 0.445, 0.230, 0.083, 0.065 and 0.050 respectively. Furthermore, the mutant haplotype (− 53(TA)7/− 3279G/− 3156A) was found to have a significant effect on bilirubin concentrations. Promoter polymorphisms and a common haplotype of the UGT1A1 gene are associated with serum bilirubin concentrations and could be a genetic risk factor for hyperbilirubinemia in Indians.     

Identification of the deletions in the UGT1A1 gene of the patients with Crigler-Najjar syndrome type I from Slovakia

Crigler-Najjar syndrome type I (CN I) is a rare autosomal recessive disorder due to hepatic dysfunction of uridine diphospho-glucuronosyltransferase (UGT) activity toward bilirubin. Complete inactivation of this enzyme causing CN I lead to accumulation of unconjugated bilirubin in serum and bile. Here we report the results of the molecular characterization of the uridine diphospho-glucuronosyltransferase 1A1 (UGT1A1) gene in a consanguineous family of Slovak Roms and an unrelated non-Romany family with CN I. Sequence analysis of UGT1A1 gene in all four Romany patients showed mutation in exon 4, a deletion of an A at codon 407 (1220delA), not yet described in homozygous status. All analysed patients were homozygous for 1220delA mutation and their 3 healthy sibs were heterozygous. The non-Romany patient was a compound heterozygote for two different deletions, 1220delA and 717-718delAG at codon 239. In the family of his cousin a son was born affected with CN I, who was homozygote for 717-718delAG mutation. His other niece affected with CN II was heterozygote for mutation 717-718delAG but homozygote for TA insertion and enhancer substitution T-3279G. Haplotype analysis suggests that the 1220delA mutation is identical by descent in both families, though they originate from two ethnically different populations (Slovaks vs. Roms).     

Genetic polymorphisms in the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene and prostate cancer risk in Caucasian men

Background: Catechol-estrogen metabolites can induce carcinogenesis by acting as endogenous tumor initiators. Glucuronidation, mediated by the UDP-glucuronosyltransferase 1A1 (UGT1A1) enzyme, is a main metabolic pathway of estrogen detoxification in steroid target tissues, such as the prostate. The aim of our study was to investigate the possible correlation between UGT1A1 promoter gene polymorphisms and prostate cancer risk. Patients and methods: 129 patients with prostate cancer and 260 healthy controls were included in our study. A(TA)TAA promoter polymorphism of UGT1A1 gene was studied using the Fragment Analysis Software of an automated DNA sequencer and three genotypes (homozygous 7/7, heterozygous 6/7 and normal homozygous 6/6) were identified. Results: No significant differences were observed between the cancer group and controls regarding the genotyping distribution of the three UGT1A1 promoter genotypes (P > 0.05). Also, no association was found between overall disease risk and the presence of the polymorphic homozygous genotype (TA(7)/TA(7) vs TA(6)/TA(7) + TA(6)/TA(6)) (P = 0.18). In addition, no association was revealed between UGT1A1 genotype distribution and Gleason score (P = 0.55). Conclusion: Our data suggest that the TA repeat polymorphism of UGT1A1 gene does not seem to alter prostate cancer risk susceptibility in Caucasian men.     

Lack of association between the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene polymorphism and the risk of benign prostatic hyperplasia in Caucasian men

Glucuronidation, mediated by the UDP-glucuronosyltransferase 1A1 (UGT1A1) enzyme, is an important metabolic process during which steroids are converted to more easily excreted compounds in steroid target tissues, such as the prostate. The aim of our study was to investigate the possible correlation between UGT1A1 promoter gene polymorphism and benign prostatic hyperplasia. 421 blood samples were obtained from 138 consecutive patients diagnosed with benign prostatic hypeplasia (BPH group) and 283 healthy volunteers (control group). A(TA)6TAA promoter polymorphism of UGT1A1 gene was studied using the Fragment Analysis Software of an automated DNA sequencer and three genotypes (homozygous 7/7, heterozygous 6/7 and normal homozygous 6/6) were identified. No significant differences were observed between the BPH group and controls regarding the genotyping distribution of the three UGT1A1 promoter genotypes (P = 0.39). Also, no association was found between overall disease risk and the presence of the polymorphic homozygous genotype (TA(7)/TA)7) vs. TA(6)/TA(7) + TA(6)/TA(6)) (P = 0.31). Our data suggest that the TA repeat polymorphism of UGT1A1 is not associated with increased BPH risk susceptibility in Caucasian men.     

Identification of a missense mutation (G329A;Arg(110)--> GLN) in the human FUT7 gene

The human FUT7 gene codes for the alpha1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLe(x)) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLe(x) and SLe(x)-related epitopes. One individual showed lower levels of SLe(x) expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg(110) --> Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. The FUT7 Arg(110) is conserved in all previously cloned vertebrate alpha 1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of < or =1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7 alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLe(x) and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sj?gren's syndrome but had no history of recurrent bacterial infections or leukocytosis.     

Identification of Promotor and Exonic Variations,and Functional Characterization of a Splice Site Mutation in Indian Patients with Unconjugated Hyperbilirubinemia

Background

Mild unconjugated hyperbilirubinemia (UH), due to reduced activity of the enzyme uridine diphosphoglucuronate-glucuronosyltransferase family, polypeptide 1 (UGT1A1), is a common clinical condition. Most cases are caused by presence in homozygous form of an A(TA)₇TAA nucleotide sequence instead of the usual A(TA)₆TAA sequence in promoter region of the UGT1A1 gene. In some cases, other genetic variations have been identified which differ between populations. There is need for more data on such genetic variations from India.

Methods

DNA from subjects with unexplained persistent or recurrent UH was tested for the presence of TA promoter insertions. In addition, all five exons and splicing site regions of UGT1A1 gene were sequenced. Several bioinformatics tools were used to determine the biological significance of the observed genetic changes. Functional analysis was done to look for effect of a splice site mutation in UGT1A1.

Results

Of 71 subjects with UH (68 male; median age [range], 26 [16–63] years; serum bilirubin 56 [26–219] μM/L, predominantly unconjugated) studied, 65 (91.5%) subjects were homozygous for A(TA)₇TAA allele, five (7.0%) were heterozygous, and one (1.4%) lacked this change. Fifteen subjects with UH had missense exonic single nucleotide changes (14 heterozygous, 1 homozygous), including one subject with a novel nucleotide change (p.Thr205Asn). Bioinformatics tools predicted some of these variations (p.Arg108Cys, p.Ile159Thr and p.Glu463Val) to be deleterious. Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein.

Conclusion

Our study revealed several single nucleotide variations in UGT1A1 gene in Indian subjects with UH. Functional characterization of a splice site variation indicated that it leads to disordered splicing. These variations may explain UH in subjects who lacked homozygous A(TA)₇TAA promoter alleles.     

一个中国Gilbert综合征家系的遗传学分析

对一个中国汉族Gilbert综合征遗传家系致病基因突变位点进行鉴定,以期了解该病的分子遗传学基础。首先提取先证者基因组DNA,PCR扩增尿苷二磷酸葡萄糖醛酸转移酶UGT1A1基因的5个外显子,以琼脂糖电泳鉴定PCR产物,纯化后直接测序鉴定。基因扫描显示,与血清胆红素水平密切相关的UGT1A1基因在第1和第5外显子存在纯合突变,而 UGT1A1基因启动子区域和内含子/外显子剪接边界位点序列未检测到突变。进一步对其他家系成员该基因的相应位点进行突变检测,结果显示他们在第1和第5外显子也存在杂合突变,其中还有两个成员在启动子区域检测到(TA)插入突变。对家系成员未抗凝新鲜血液进行生化检测证实了基因突变分析的结果。综合以上结果发现该家系三种突变并存,致病因素为第1和/或第5外显子突变,为显性遗传,两种突变位点纯合导致先证者出现严重胆红素代谢功能障碍。该家系因此成为Gilbert综合征突变位点及其致病机理研究的一个典型临床病例。     

Novel splice, missense, and nonsense mutations in the fumarylacetoacetase gene causing tyrosinemia type 1.

In six unrelated patients with hereditary tyrosinemia type 1 (HT1), three different disease-causing mutations were found by DNA sequencing. Two Pakistani patients, with acute and intermediate forms of HT1, were homozygous for a G192-->T mutation in the last nucleotide of exon 2. This caused aberrant splicing with partial intron 2 retention and premature termination. Three Turkish patients with chronic and intermediate forms of HT1 were homozygous for an A698-->T mutation substituting aspartic acid 233 with valine. A Norwegian patient with an intermediate clinical phenotype was heterozygous for G786-->A, introducing a TGA stop codon for Trp262 (W262X). Site-directed mutagenesis and expression in a rabbit reticulocyte lysate system demonstrated that the nonsense and missense mutations abolished fumarylacetoacetase activity and gave reduced amounts of a truncated and a full-length protein, respectively. Simple tests were established to identify the three mutations by restriction digestion of PCR-amplified genomic DNA. Among 30 additional HT1 patients investigated, 2 were found to be homozygous and 1 heterozygous for G192-->T. Two other patients were homozygous and one was heterozygous for W262X.     

A polymorphism in the human cytotoxic T-lymphocyte antigen 4 (<Emphasis Type="Italic">CTLA4</Emphasis>) gene (exon 1 +49) alters T-cell activation

The cytotoxic T-lymphocyte antigen 4 (CTLA4) is an important modifier of T-cell activation with down-regulatory properties upon B7 engagement. An allelic polymorphism in exon 1 of the CTLA4 gene coding for the peptide leader sequence of CTLA4 was recently described. This polymorphism was detected in association with several autoimmune diseases. In this study, we investigated the functional impact of the CTLA4 exon 1 +49 A/G dimorphism on T-cell activation and cellular localization. We examined the T-cell response from healthy donors either homozygous for A or G at position +49 of the exon 1. Under suboptimal stimulation conditions we found a greater proliferative response of cells from donors homozygous for G at position +49. FACS analysis of CTLA4 expression revealed a reduced up-regulation of CTLA4 from G/G donors upon T-cell activation, if compared with wild-type cells. Intracellular CTLA4 distribution demonstrated qualitatively different staining patterns between the two genotypes as determined using confocal fluorescence microscopy. Our results suggest that the G allele at position +49 of exon 1 affects the CTLA4-driven down-regulation of T-cell activation and may be an important factor in the pathogenesis of autoimmune diseases.     

NPC1 gene mutations in Japanese patients with Niemann-Pick disease type C

Complementary and genomic DNAs isolated from the fibroblasts of 10 Japanese (7 late infantile, 2 juvenile, and 1 adult form of the disease) and one Caucasian patient with Niemann-Pick disease type C were analyzed for mutations in the NPC1 gene. Fourteen novel mutations were found including small deletions and point mutations. A one-base deletion and a point mutation caused splicing errors. The mutations were not clustered in any particular region of the gene and were found both in and out of the transmembrane domains. Three patients were homozygous, five were compound heterozygous, and the remaining three were suspected of being compound hetrozygous with an unknown error in one of their NPC1 alleles. Of the 14 mutations, the G1553A substitution that caused a splicing error of exon 9 appeared to be relatively common in Japanese patients, because two patients were homozygous and one patient was compound heterozygous for this mutation. Electronic Publication     

Splice-site mutations: a novel genetic mechanism of Crigler-Najjar syndrome type 1.

Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression.     

Spectrum and frequency of mutations in the connexin 32 gene (<Emphasis Type="Italic">GJB1</Emphasis>) in hereditary and sensory neuropathy type 1X patients from Bashkortostan

Hereditary motor and sensory neuropathy type 1X (HMSN 1X) is the second most frequent form of demyelinating polyneuropathies and is caused by mutations in the gene for connexin 32 protein (Cx32, GJB1). The contribution of HMSN 1X to the structure of HMSN in the Republic of Bashkortostan was determined. The GJB1 mutations were detected in 18 out of 131 unrelated patients, which constituted 13.7%. The four missense mutations identified were represented by: p.Pro87Ala (c.259C > G) with the frequency of 10%; p.Arg22Gln (c.65G > A) (2.98%); p.Arg15Gln (c.44G > A); and p.Thr86Ile (c.257C > T) (0.8%). The latter mutation was never described before. The frequent mutation p.Pro87Ala was tested for linkage disequilibrium with the alleles of five polymorphic microsatellite DNA loci associated with the GJB1. It was demonstrated that 10 out of 13 chromosomes carrying the mutation mentioned had common DXS8111-DXS983-DXS8107-DXS8052 haplotype. This finding suggested the distribution of this mutation on the territory of the Republic of Bashkortostan as a result of the founder effect. The mutational spectrum of GJB1 and mutation frequencies observed in the HMSN 1X patients examined were characterized by ethnic heterogeneity. This finding will provide development of most optimal algorithm of the HMSN DNA diagnostics in the region.     

Genotype frequencies of UDP-glucuronosyltransferase 1A1 promoter gene polymorphism in the population of healthy Croatian pre-scholars

Increased serum bilirubin levels in patients with Gilbert syndrome (GS) are caused by reduction of hepatic activity of bilirubin glucuronosyltranferase to about 30% of normal. UGT1A1 genetic polymorphism with absent or very low bilirubin UDP-glucuronosyltransferase (B-UGT) activity is associated with Gilbert's syndrome (GS) and other hyperbilirubinemias. The genetic basis of GS is the insertion of two additional TA nucleotides (resulting in seven repeats of TA) in the TATAA box, present in proximal promoter of UGT1A1 gene. This study included 323 Croatian pre-scholars, including 164 boys and 159 girls. Statistical analysis showed significant difference for total bilirubin concentration between different genotypes (p < 0.001). Also, statistically significant difference for total bilirubin concentration was emphasized between genotypes 6/6 and 7/7 (p < 0.001) as well as 6/7 and 7/7 (p < 0.001). Higher total plasma bilirubin concentrations are significantly correlated with 7/7 genotype which is present in 9.8% of population studied.     

Identical G+1 to A mutations in three different introns of the type III procollagen gene (COL3A1) produce different patterns of RNA splicing in three variants of Ehlers-Danlos syndrome. IV. An explanation for exon skipping some mutations and not others

Identical G+1 mutations in three different introns of the gene for type III procollagen (COL3A1) that cause aberrant splicing of RNA were found in three probands with life-threatening variants of Ehlers-Danlos syndrome. Because the three mutations were in a gene with multiple and homologous exons, they provided an interesting test for factors that influence aberrant splicing. The G+1 to A mutation in intron 16 caused extensive exon skipping, the G+1 to A mutation in intron 20 caused both use of a cryptic splice site and retention of all the intron sequences, and the G+1 to A mutation in intron 42 caused efficient use of a single cryptic splice site. The different patterns of RNA splicing were not explained by evaluation of potential cryptic splice sites in the introns by either their homology with 5'-splice sites from other genes or by their delta G(0)37 values for binding to U1 RNA. Instead, the results suggested that the patterns of aberrant RNA splicing were primarily determined by the relative rates at which adjacent introns were normally spliced.     

Simultaneous enhancement of thermostability and catalytic activity of phospholipase A(1) by evolutionary molecular engineering

The thermal stability and catalytic activity of phospholipase A(1) from Serratia sp. strain MK1 were improved by evolutionary molecular engineering. Two thermostable mutants were isolated after sequential rounds of error-prone PCR performed to introduce random mutations and filter-based screening of the resultant mutant library; we determined that these mutants had six (mutant TA3) and seven (mutant TA13) amino acid substitutions. Different types of substitutions were found in the two mutants, and these substitutions resulted in an increase in nonpolar residues (mutant TA3) or in differences between side chains for polar or charged residues (mutant TA13). The wild-type and mutant enzymes were purified, and the effect of temperature on the stability and catalytic activity of the enzymes was investigated. The melting temperatures of the TA3 and TA13 enzymes were increased by 7 and 11 degrees C, respectively, compared with the melting temperature of the wild-type enzyme. Thus, we found that evolutionary molecular engineering was an effective and efficient approach for increasing thermostability without compromising enzyme activity.     

A homozygous <Emphasis Type="Italic">RAB3GAP2</Emphasis> mutation causes Warburg Micro syndrome

Warburg Micro syndrome and Martsolf syndrome are clinically overlapping autosomal recessive conditions characterized by congenital cataracts, microphthalmia, postnatal microcephaly, and developmental delay. The neurodevelopmental and ophthalmological phenotype is more severe in Warburg Micro syndrome in which cerebral malformations and severe motor and mental retardation are common. While biallelic loss-of-function mutations in RAB3GAP1 are present in the majority of patients with Warburg Micro syndrome; a hypomorphic homozygous splicing mutation of RAB3GAP2 has been reported in a single family with Martsolf syndrome. Here, we report a novel homozygous RAB3GAP2 small in-frame deletion, c.499_507delTTCTACACT (p.Phe167_Thr169del) that causes Warburg Micro syndrome in a girl from a consanguineous Turkish family presenting with congenital cataracts, microphthalmia, absent visually evoked potentials, microcephaly, polymicrogyria, hypoplasia of the corpus callosum, and severe developmental delay. No RAB3GAP2 mutations were detected in ten additional unrelated patients with RAB3GAP1-negative Warburg Micro syndrome, consistent with further genetic heterogeneity. In conclusion, we provide evidence that RAB3GAP2 mutations are not specific to Martsolf syndrome. Rather, our findings suggest that loss-of-function mutations of RAB3GAP1 as well as functionally severe RAB3GAP2 mutations cause Warburg Micro syndrome while hypomorphic RAB3GAP2 mutations can result in the milder Martsolf phenotype. Thus, a phenotypic severity gradient may exist in the RAB3GAP-associated disease continuum (the “Warburg–Martsolf syndrome”) which is presumably determined by the mutant gene and the nature of the mutation.     

Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type 1A.

Carbohydrate-deficient-glycoprotein syndrome type 1 (CDG1; also known as "Jaeken syndrome") is an autosomal recessive disorder characterized by defective glycosylation. Most patients show a deficiency of phosphomannomutase (PMM), the enzyme that converts mannose 6-phosphate to mannose 1-phosphate in the synthesis of GDP-mannose. The disease is linked to chromosome 16p13, and mutations have recently been identified in the PMM2 gene in CDG1 patients with a PMM deficiency (CDG1A). The availability of the genomic sequences of PMM2 allowed us to screen for mutations in 56 CDG1 patients from different geographic origins. By SSCP analysis and by sequencing, we identified 23 different missense mutations and 1 single-base-pair deletion. In total, mutations were found on 99% of the disease chromosomes in CDG1A patients. The R141H substitution is present on 43 of the 112 disease alleles. However, this mutation was never observed in the homozygous state, suggesting that homozygosity for these alterations is incompatible with life. On the other hand, patients were found homozygous for the D65Y and F119L mutations, which must therefore be mild mutations. One particular genotype, R141H/D188G, which is prevalent in Belgium and the Netherlands, is associated with a severe phenotype and a high mortality. Apart from this, there is only a limited relation between the genotype and the clinical phenotype.