Fabry disease: twenty novel alpha-galactosidase A mutations and genotype-phenotype correlations in classical and variant phenotypes

BACKGROUND: Fabry disease (OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism resulting from mutations in the alpha-galactosidase A (alpha-Gal A) gene. The disease is phenotypically heterogeneous with classic and variant phenotypes. To assess the molecular heterogeneity, define genotype/phenotype correlations, and for precise carrier identification, the nature of the molecular lesions in the alpha-Gal A gene was determined in 40 unrelated families with Fabry disease. MATERIALS AND METHODS: Genomic DNA was isolated from affected males or obligate carrier females and the entire alpha-Gal A coding region and flanking sequences were amplified by PCR and analyzed by automated sequencing. Haplotype analyses were performed with polymorphisms within and flanking the alpha-Gal A gene. RESULTS: Twenty new mutations were identified (G43R, R49G, M72I, G138E, W236X, L243F, W245X, S247C, D266E, W287C, S297C, N355K, E358G, P409S, g1237del15, g10274insG, g10679insG, g10702delA, g11018insA, g11185-delT), each in a single family. In the remaining 20 Fabry families, 18 previously reported mutations were detected (R49P, D92N, C94Y, R112C [two families], F113S, W162X, G183D, R220X, R227X, R227Q, Q250X, R301X, R301Q, G328R, R342Q, E358K, P409A, g10208delAA [two families]). Haplotype analyses indicated that the families with the R112C or g10208delAA mutations were not related. The proband with the D266E lesion had a severe classic phenotype, having developed renal failure at 15 years. In contrast, the patient with the S247C mutation had a variant phenotype, lacking the classic manifestations and having mild renal involvement at 64 years. CONCLUSIONS: These results further define the heterogeneity of alpha-Gal A mutations causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis in these families, and provide additional genotype/phenotype correlations in this lysosomal storage disease.     

Mutations in the HFE, TFR2, and SLC40A1 genes in patients with hemochromatosis

Hereditary hemochromatosis causes iron overload and is associated with a variety of genetic and phenotypic conditions. Early diagnosis is important so that effective treatment can be administered and the risk of tissue damage avoided. Most patients are homozygous for the c.845G>A (p.C282Y) mutation in the HFE gene; however, rare forms of genetic iron overload must be diagnosed using a specific genetic analysis. We studied the genotype of 5 patients who had hyperferritinemia and an iron overload phenotype, but not classic mutations in the HFE gene. Two patients were undergoing phlebotomy and had no iron overload, 1 with metabolic syndrome and no phlebotomy had mild iron overload, and 2 patients had severe iron overload despite phlebotomy. The patients' first-degree relatives also underwent the analysis. We found 5 not previously published mutations: c.-408_-406delCAA in HFE, c.1118G>A (p.G373D), c.1473G>A (p.E491E) and c.2085G>C (p.S695S) in TFR2; and c.-428_-427GG>TT in SLC40A1. Moreover, we found 3 previously published mutations: c.221C>T (p.R71X) in HFE; c.1127C>A (p.A376D) in TFR2; and c.539T>C (p.I180T) in SLC40A1. Four patients were double heterozygous or compound heterozygous for the mutations mentioned above, and the patient with metabolic syndrome was heterozygous for a mutation in the TFR2 gene. Our findings show that hereditary hemochromatosis is clinically and genetically heterogeneous and that acquired factors may modify or determine the phenotype.     

Interaction of hemojuvelin with neogenin results in iron accumulation in human embryonic kidney 293 cells

Type 2 hereditary hemochromatosis (HH) or juvenile hemochromatosis is an early onset, genetically heterogeneous, autosomal recessive disorder of iron overload. Type 2A HH is caused by mutations in the recently cloned hemojuvelin gene (HJV; also called HFE2) (Papanikolaou, G., Samuels, M. E., Ludwig, E. H., MacDonald, M. L., Franchini, P. L., Dube, M. P., Andres, L., MacFarlane, J., Sakellaropoulos, N., Politou, M., Nemeth, E., Thompson, J., Risler, J. K., Zaborowska, C., Babakaiff, R., Radomski, C. C., Pape, T. D., Davidas, O., Christakis, J., Brissot, P., Lockitch, G., Ganz, T., Hayden, M. R., and Goldberg, Y. P. (2004) Nat. Genet. 36, 77-82), whereas Type 2B HH is caused by mutations in hepcidin. HJV is highly expressed in both skeletal muscle and liver. Mutations in HJV are implicated in the majority of diagnosed juvenile hemochromatosis patients. In this study, we stably transfected HJV cDNA into human embryonic kidney 293 cells and characterized the processing of HJV and its effect on iron homeostasis. Our results indicate that HJV is a glycosylphosphatidylinositol-linked protein and undergoes a partial autocatalytic cleavage during its intracellular processing. HJV co-immunoprecipitated with neogenin, a receptor involved in a variety of cellular signaling processes. It did not interact with the closely related receptor DCC (deleted in Colon Cancer). In addition, the HJV G320V mutant implicated in Type 2A HH did not co-immunoprecipitate with neogenin. Immunoblot analysis of ferritin levels and transferrin-55Fe accumulation studies indicated that the HJV-induced increase in intracellular iron levels in human embryonic kidney 293 cells is dependent on the presence of neogenin in the cells, thus linking these two proteins to intracellular iron homeostasis.     

Uroporphyrinogen decarboxylase: complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria.

A deficiency in uroporphyrinogen decarboxylase (UROD) enzyme activity, the fifth enzyme of the heme biosynthetic pathway, is found in patients with sporadic porphyria cutanea tarda (s-PCT), familial porphyria cutanea tarda (f-PCT), and hepatoerythropoietic porphyria (HEP). Subnormal UROD activity is due to mutations of the UROD gene in both f-PCT and HEP, but no mutations have been found in s-PCT. Genetic analysis has determined that f-PCT is transmitted as an autosomal dominant trait. In contrast, HEP, a severe form of cutaneous porphyria, is transmitted as an autosomal recessive trait. HEP is characterized by a profound deficiency of UROD activity, and the disease is usually manifest in childhood. In this study, a strategy was designed to identify alleles responsible for the HEP phenotype in three unrelated families. Mutations of UROD were identified by direct sequencing of four amplified fragments that contained the entire coding sequence of the UROD gene. Two new missense mutations were observed at the homoallelic state: P62L (proline-to-leucine substitution at codon 62) in a Portuguese family and Y311C (tyrosine-to-cysteine substitution at codon 311) in an Italian family. A third mutation, G281E, was observed in a Spanish family. This mutation has been previously described in three families from Spain and one from Tunisia. In the Spanish family described in this report, a paternal uncle of the proband developed clinically overt PCT as an adult and proved to be heterozygous for the G281E mutation. Mutant cDNAs corresponding to the P62L and Y311C changes detected in these families were created by site-directed mutagenesis. Recombinant proteins proved to have subnormal enzyme activity, and the Y311C mutant was thermolabile.     

Heterogeneous mutations in the glycogen-debranching enzyme gene are responsible for glycogen storage disease type IIIa in Japan

Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disorder caused by deficiency of the glycogen-debranching enzyme (AGL). Recent studies of the AGL gene have revealed the prevalent mutations in North African Jewish and Caucasian populations, but whether these common mutations are present in other ethnic groups remains unclear. We have investigated eight Japanese GSD IIIa patients from seven families and identified seven mutations, including one splicing mutation (IVS 14+1G-->T) previously reported by us, together with six novel ones: a nonsense mutation (L124X), a splice site mutation (IVS29-1G-->C), a 1-bp deletion (587delC), a 2-bp deletion (4216-4217delAG), a 1-bp insertion (2072-2073insA), and a 3-bp insertion (4735-4736insTAT). The last mutation results in insertion of a tyrosine residue at a putative glycogen-binding site, and the rest are predicted to cause synthesis of truncated proteins lacking the glycogen-binding site at the carboxyl terminal. Thirteen novel polymorphisms have also been revealed in this study: three amino acid substitutions (R387Q, G1115R, and E1343 K), one silent point mutation (L298L), one nucleotide change in the 5'-noncoding region, and eight nucleotide changes in introns. Haplotype analysis with combinations of these polymorphic markers showed L124X, IVS14+1G-->T, and 4216-4217delAG to be on different haplotypes. These results demonstrate the importance of the integrity of the carboxy terminal domain in the AGL protein and the molecular heterogeneity of GSD IIIa in Japan.     

Characterization of three novel pathogenic SLC40A1 mutations and genotype/phenotype correlations in 7 Italian families with type 4 hereditary hemochromatosis

Mutations of SLC40A1 encoding ferroportin (Fpn), the unique cellular iron exporter, severely affect iron homeostasis causing type 4 hereditary hemochromatosis, an autosomal dominant iron overload condition with variable phenotypic manifestations. This disease can be classified as type 4A, better known as “ferroportin disease”, which is due to “loss of function” mutations that lead to decreased iron export from cells, or as type 4B hemochromatosis, which is caused by “gain of function” mutations, conferring partial or complete resistance to hepcidin-mediated Fpn degradation.In this work, we discuss clinical and molecular findings on a group of patients in whom a SLC40A1 single copy missense variant was identified. Three novel variants, p.D181N, p.G204R and p.R296Q were functionally characterized. Fpn D181N and R296Q mutants can be classified as full or partial loss of function, respectively. Replacement of G204 with arginine appears to cause a more complex defect with impact both on iron export function and hepcidin sensitivity. This finding confirms the difficulty of predicting the effect of a mutation on the molecular properties of Fpn in order to provide an exhaustive explanation to the wide variability of the phenotype in type 4 hereditary hemochromatosis.     

Novel and recurrent mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia

We have identified 16 different mutations of the low-density lipoprotein receptor (LDLR) gene in 25 unrelated Korean patients with heterozygous familial hypercholesterolemia (FH), including five novel mutations, C83Y, 661del17, 1705insCTAG, C675X, and 941-1G>A. The 1705insCTAG mutation in which the four 3 cent -terminal nucleotides of exon 11 are duplicated was found to prevent splicing of exon 11 and would therefore generate a truncated polypeptide. The in-frame 36-bp deletion (1591del36) in exon 11, which had been reported only in one Korean FH patient, was also found. We showed that this change affects transport of the LDL receptor from the endoplasmic reticulum to the cell surface. In addition, we found 8 mutations (-136C>T, E119K, E207K, E207X, F382L, R574Q, 1846-1G>A, and P664L) that had been described in other ethnic groups but not in Koreans, and 2 mutations (R94H and D200N) that had been described in Koreans as well as other ethnic groups. 5 mutations (1591del36, E119K, E207X, E207K, and P664L) were found more than once in the Korean FH samples. Identification of the novel and recurring LDLR mutations in Korean FH patients should facilitate prenatal and early diagnosis in families at high risk of FH.     

Disease-causing Mutations in Exon 11 of the Medium-Chain Acyl-CoA Dehydrogenase Gene

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial β-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985), causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, all of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been suggested that this exon may be a mutational hot spot. Here we describe the results from sequence analysis of exon 11 and part of the flanking introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11. Our results show that exon 11 is not especially mutation prone. We demonstrate that two of the identified disease-causing mutations can be detected by restriction enzyme digestion of the PCR product from the assay for the G985 mutation, a discovery that makes this assay even more useful than before. On the basis of expression of wild-type and mutant MCAD protein in COS-7 cells, we show that the identified mutations abolish MCAD enzyme activity and that they therefore must be disease causing. The M301T, S311R, and K304E mutations are located in helix H, which makes up part of the dimer-dimer interface of the MCAD tetramer. On the basis of the three-dimensional structure of MCAD and the results from the COS-7 expression experiments, we speculate that the primary effect of the M301T and S311R mutations is on correct folding/tetramer assembly, as it has previously been observed for the K304E mutation.     

Detection of common mutations in the GALT gene through ARMS

Type I galactosemia is an inborn error resulting from mutations on both alleles of the GALT gene, which leads to the absence or deficiency of galactose-1-phosphate uridyltranseferase (GALT), the second of three enzymes catalyzing the conversion of galactose into glucose. On the basis of residual GALT activity, Type I galactosemia is classified into severe “Classical” and mild “Duarte” phenotypes. Classical galactosemia is frequently associated with S135L, Q188R and K285N mutations in the GALT gene. The functionally neutral N314D variation in the GALT gene is associated with Duarte galactosemia and is widespread among various worldwide populations. The present study aimed at detecting S135L, Q188R and K285N mutations and the N314D variant in the GALT gene by PCR using amplification refractory mutation system (ARMS). ARMS assays were established using standard DNA samples and were used for 8 galactosemia patients and 190 unrelated normal subjects all of Pakistani origin. S135L and K285N mutations were present neither in galactosemia patients nor in normal subjects. Only one galactosemia patient carried Q188R mutation that was in homozygous state. However, the N314D variant was frequently found both in affected (7 out of 16 alleles) and normal subjects (55 out of 380 alleles). This finding indicates that Duarte allele D314 might be far more common in Pakistani population than in European and North American ones.     

The identification of a domain in Escherichia coli elongation factor Tu that interacts with elongation factor Ts.

A method has been developed to search for the elongation factor Tu (EF-Tu) domain(s) that interact with elongation factor Ts (EF-Ts). This method is based on the suppression of Escherichia coli EF-Tu-dominant negative mutation K136E, a mutation that exerts its effect by sequestering EF-Ts. We have identified nine single-amino acid- substituted suppression mutations in the region 146-199 of EF-Tu. These mutations are R154C, P168L, A174V, K176E, D181G, E190K, D196G, S197F, and I199V. All suppression mutations but one (R154C) significantly affect EF-Tu's ability to interact with EF-Ts under equilibrium conditions. Moreover, with the exception of mutation A174V, the GDP affinity of EF-Tu appears to be relatively unaffected by these mutations. These results suggest that the domain of residues 154 to 199 on EF-Tu is involved in interacting with EF-Ts. These suppression mutations are also capable of suppressing dominant negative mutants N135D and N135I to various degrees. This suggests that dominant negative mutants N135D and N135I are likely to have the same molecular basis as the K136E mutation. The method we have developed in this study is versatile and can be readily adapted to map other regions of EF-Tu. A model of EF-Ts-catalyzed guanine-nucleotide exchange is discussed.     

Spectrum of cystic fibrosis mutations in Serbia and Montenegro and strategy for prenatal diagnosis

We have screened 175 patients for molecular defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using nondenaturing polyacrylamide gel electrophoresis (PAGE), denaturing gradient gel electrophoresis (DGGE), and sequencing. Six different mutations (F508del, G542X, 621+1G --> T, 2789+5G --> A, R1070Q, and S466X) accounted for 79.71% of CF alleles, with the F508del mutation showing a frequency of 72.28%. Another 12 mutations (R334W, 2184insA, I507del, 1525-1G --> A, E585X, R75X, M1I, 457TAT --> G, 574delA, 2723delTT, A120T, and 2907delTT) covered an additional 3.36%. A novel mutation (2723delTT) was found in one CF patient (F508del/2723delTT). Thus, a total of 18 mutations cover 82.57% of CF alleles. During our study, 72% of families at risk for having a CF child were found to be fully informative for prenatal diagnosis. Prenatal diagnosis was performed on 56 families; 76 analyses resulting in 16 affected, 38 carriers, and 22 healthy fetuses. These results imply that the molecular basis of CF in Serbia and Montenegro is highly heterogeneous, as is observed in other eastern and southern European populations. Because we detected more then 80% of CFTR alleles, results could be used for planning future screening and appropriate genetic counseling programs in our country.     

Prevalence of widespread BRCA1 gene mutations in patients with familial breast cancer from St. Petersburg

Ten variants different from the canonical nucleotide sequence (GenBank, U14680) has been identified when studying the mutation spectrum in gene BRCA1. Six of them (5382insC, 2963del10, 3819de15, 3875del4, 2274insA, and R1203X) cause premature termination of protein synthesis, thus predisposing to breast cancer. A missense mutation E1250K is presumed to be a factor of predisposition to cancer. We classified three variants of nucleotide sequence found in some patients as DNA polymorphisms S694S, L771L, and E1038G. The 5382insC and 3819de15 mutations have been detected in four and two families, respectively. Five of the mutations detected have not been found in Russia before. However, all mutations except for 2963del10 have been found in other populations of the world, which indicates their long evolutionary history. Two mutations found in patients from St. Petersburg (5382insC and 3875de14) have also been found in oncological patients from other regions of the Russian Federation.     

A primer for predicting risk of disease in HFE-linked hemochromatosis

Since the discovery of the hemochromatosis gene (HFE) in 1996, there has been increasing interest in diagnostic testing for the C282Y and H63D mutations. The high frequency of these two alleles and their incomplete penetrance in homozygotes and compound heterozygotes make genetic counseling for hemochromatosis different from some other autosomal recessive conditions in that parents and children may also be at risk for iron overload, while homozygotes may remain asymptomatic. We provide a guideline for genetic counseling in HFE-linked hemochromatosis based on the genetic probability of inheriting HFE mutations and known information about expression of iron overload in various HFE genotypes. Genetic probabilities were based on allele frequencies derived from large population studies and Hardy-Weinberg equilibrium estimates. Expression of iron overload in those of various genotypes was based on available estimates of serum ferritin from population screening studies. Estimates for the likelihood of clinical iron overload requiring follow-up screening or treatment are provided by gender and genotype. The probability of inheriting HFE mutations and developing iron overload can be estimated in family members of a proband with HFE mutations. Many C282Y homozygotes will not have clinical iron overload. The risk is highest in men and their C282Y homozygous brothers and significantly lower in homozygous women. Iron overload is uncommon in compound heterozygotes and H63D homozygotes.     

Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background

Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis.

Methods

The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed.

Results

We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury.

Conclusion

In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.     

Active site mutations of recombinant deacetoxycephalosporin C synthase

Site-directed mutagenesis of active site residues of deacetoxycephalosporin C synthase active site residues was carried out to investigate their role in catalysis. The following mutations were made and their effects on the conversion of 2-oxoglutarate and the oxidation of penicillin N or G were assessed: M180F, G299N, G300N, Y302S, Y302F/G300A, Y302E, Y302H, and N304A. The Y302S, Y302E, and Y302H mutations reduced 2-oxoglutarate conversions and abolished (<2%) penicillin G oxidation. The Y302F/G300A mutation caused partial uncoupling of penicillin G oxidation from 2-oxoglutarate conversion, but did not uncouple penicillin N oxidation from 2-oxoglutarate conversion. Met-180 is involved in binding 2-oxoglutarate, and the M180F mutation caused uncoupling of 2-oxoglutarate from penicillin oxidation. The N304A mutation apparently enhanced in vitro conversion of penicillin N but had little effect on the oxidation of penicillin G, under standard assay conditions.     

Novel sodium channel gene mutations in Blattella germanica reduce the sensitivity of expressed channels to deltamethrin

Pyrethroid insecticides alter the normal gating of voltage-gated sodium channels in the nervous system. Three sodium channel mutations (E434K, C764R, L993F) were recently identified in pyrethroid resistant German cockroach populations. In this report, we show that the L993F mutation decreased sodium channel sensitivity to the pyrethroid, deltamethrin, by five-fold in Xenopus oocytes. In contrast, neither E434K nor C764R alone decreased channel sensitivity to deltamethrin. However, E434K or C764R combined with L993F reduced deltamethrin sensitivity by 100-fold. Furthermore, concomitant presence of all three mutations (KRF) reduced channel sensitivity to deltamethrin by 500-fold. None of the mutations significantly affected channel gating. However, sodium current amplitudes from the mutant sodium channel carrying either E434K or C764R alone were much reduced compared to those of the wild-type channel or the channel carrying the double or triple mutations (KF, RF and KRF). These results indicated that evolution of sodium channel insensitivity in the German cockroach is achieved by sequential selection of a primary mutation L993F and two secondary mutations E434K and C764R, and concomitant presence of all three mutations dramatically reduced sodium channel sensitivity to deltamethrin.     

Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation

The specific functions of the amino acid residues in the streptokinase (SK) gamma-domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK gamma-domain, SK(K278A,K279A,E281A,K282A), and SK(D360A,R363A) had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SK(D322A,R324A,D325A) and SK(R330A,D331A,K332A,K334A) had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SK(D328A,R330A) had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg(324), Asp(325), Lys(332), and Lys(334) play important roles in the formation of a HPlg.SK complex. On the other hand, amino acid residues Asp(322), Asp(328), and Arg(330) of SK are involved in the virgin enzyme induction. Potential contact between Lys(332) of SK and Glu(623) of human microplasmin and strong interactions between Asp(328) and Lys(330), Asp(331) and Lys(334), and Asp(322) and Lys(334) of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK gamma-domain, SK(Leu(314)-Ala(342)), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.     

Frequency of MEFV gene mutations in Hatay province,Mediterranean region of Turkey and report of a novel missense mutation (I247V)

In the present study, 1000 patients with clinical suspicion of FMF were retrospectively reviewed to determine the spectrum of MEFV gene mutations by using DNA sequence analysis between September, 2008 and April, 2012. Sixteen different mutations and 55 different genotypes were detected in 618 of 1000 patients. Among 16 different mutations, R202Q (21.35%) was the most frequently observed mutation; followed by E148Q (8.85%), M694V (7.95%), M680I (2.40%), V726A (1.85%), M694I (0.95%), A744S (0.80%), R761H (0.55%), P283L (0.35%), K695R (0.20%), E230K (0.15%), L110P (0.10%), I247V (0.05%), G196W (0.05%) and G304R (0.05%). In the present study, a novel missense mutation (I247V) and a silent variant (G150G) were identified in the MEFV gene. On the other hand, P238L, G632A and G304R mutations are the first cases reported from Turkey. Our results indicated that MEFV mutations are highly heterogeneous in our study population as in other regions of Turkey and mutation screening techniques such as PCR-RFLP, amplification refractory mutation system or reverse hybridization do not adequately detect uncommon or novel mutations. Therefore, it was proven that sequence analysis of the MEFV gene could be useful for detection of rare or unknown mutations.     

Oxidative damage in colon and mammary tissue of the HFE-knockout mouse

The HFE mutation is common and, when homozygous, can lead to a morbid accumulation of body iron and the disease hereditary hemochromatosis. Heterozygotes compose 10-15% of the European-American population, and have evidence of elevated body iron compared to homozygous normal people. Dietary iron content was hypothesized to interact with the HFE genotype to influence oxidative damage in mammary and colon tissue. Two groups of HFE-knockout mice were fed a standard iron diet (300 ppm) or a low iron diet (30 ppm). There was a significantly elevated concentration of malondialdehyde (by HPLC) in mammary (305 pmol/g vs. 166, p =.04) and colon (349 pmol/g vs. 226, p =.02) tissue among those mice on the standard iron diet compared to those on the low iron diet. These results suggest that dietary modification may affect the course of iron overload from HFE mutations.     

Mutations in argininosuccinate synthetase mRNA of Japanese patients,causing classical citrullinemia.

Citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase. In order to characterize mutations in Japanese patients with classical citrullinemia, RNA isolated from 10 unrelated patients was reverse-transcribed, and cDNA amplified by PCR was cloned and sequenced. The 10 mutations identified included 6 missense mutations (A118T, A192V, R272C, G280R, R304W, and R363L), 2 mutations associated with an absence of an exon 7 or exon 13, 1 mutation with a deletion of the first 7 bp in exon 16 (which might be caused by abnormal splicing), and 1 mutation with an insertion of 37 bp within exons 15 and 16 in cDNA. The insertion mutation and the five missense mutations (R304W being excluded) are new mutations described in the present paper. These are in addition to 14 mutations (9 missense mutations, 4 mutations associated with an absence of an exon in mRNA, and 1 splicing mutation) that we identified previously in mainly American patients with neonatal citrullinemia. Two of these 20 mutations, a deletion of exon 13 sequence and a 7-bp deletion in exon 16, were common to Japanese and American populations from different ethnic backgrounds; however, other mutations were unique to each population. Furthermore, the presence of a frequent mutation--the exon 7 deletion mutation in mRNA, which accounts for 10 of 23 affected alleles--was demonstrated in Japanese citrullinemia. This differs from the situation in the United States, where there was far greater heterogeneity of mutations.