Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the gene encoding the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-Gal A; GLA). In two unrelated classically affected males, two alpha-Gal A missense mutations were identified: R112C + D313Y (c.334C>T + c.937G>T) and C172G + D313Y (c.514T>G + c.937G>T). The D313Y lesion was previously identified in classically affected males as the single mutation [Eng et al., 1993] or in cis with another missense mutation, D313Y + G411D (c.937G>T + c.1232G>A) [Guffon et al., 1998]. To determine whether the D313Y mutation was a deleterious mutation or a coding region sequence variant, the frequency of D313Y in normal X-chromosomes, as well as its enzymatic activity and subcellular localization in COS-7 cells was determined. D313Y occurred in 0.45% of 883 normal X-chromosomes, while the R112C, C172G, and G411D missense mutations were not detected in over 500 normal X-chromosomes. Expression of D313Y in COS-7 cells resulted in approximately 60% of wild-type enzymatic activity and showed lysosomal localization, while R112C, C172G, G411D, and the double-mutated constructs had markedly reduced or no detectable activity and were all retained in the endoplasmic reticulum. The expressed D313Y enzyme was stable at lysosomal pH (pH 4.6), while at neutral pH (pH 7.4), it had decreased activity. A molecular homology model of human alpha-Gal A, based on the X-ray crystal structure of chicken alpha-galactosidase B (alpha-Gal B; alpha-N-acetylgalactosaminidase) was generated [Garman et al., 2002], which provided evidence that D313Y did not markedly disrupt the alpha-Gal A enzyme structure. Thus, D313Y is a rare exonic variant with about 60% of wild-type activity in vitro and reduced activity at neutral pH, resulting in low plasma alpha-Gal A activity.     

Fabry disease: 45 novel mutations in the alpha-galactosidase A gene causing the classical phenotype

The nature of the molecular lesions in the alpha-galactosidase A (alpha-Gal A) gene causing Fabry disease was determined in 50 unrelated families with the classic phenotype of this X-linked recessive lysosomal storage disease. Genomic DNA was isolated from affected males or obligate carrier females, and the entire alpha-Gal A coding region as well as the flanking and intronic sequences were analyzed by PCR amplification and automated sequencing. Forty-five new mutations were identified including 38 single base substitutions (32 missense and four nonsense) and nine gene rearrangements: MIR, M42T, G43D, G43V, H46Y, F50C, L68F, G132R, T141I, Y152X, K168R, G183S, V199M, P205R, Y207S, Q221X, C223R, C223Y, D234Y, G271C, A288P, P293A, R301G, I303N, I317T, E341D, P362L, R363C, R363H, G373D, I384N, T385P, Q396X, E398K, S401X, P409A, g7325insC, g7384del13, g8341delG, g8391del4/ins3, g10511delTAGT, g10704delACAG, g11019insG, g11021insG, and g11048delAGG. In the remaining five Fabry families, four previously reported mutations were detected (W81X, R112C, g11011delTC, and g11050delGAG) of which the R112C substitution was found in two families who were unrelated by haplotyping. These studies further define the heterogeneity of mutations in the alpha-Gal A gene causing the classical Fabry disease phenotype, and permit precise carrier detection and prenatal diagnosis in these families.     

Fabry disease: twenty novel α-galactosidase A mutations causing the classical phenotype

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal exoglycohydrolase, alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A). The nature of the molecular lesions in the alpha-Gal A gene in 40 unrelated families with the classical phenotype (absent alpha-Gal A activity) was determined in order to provide precise heterozygote detection and prenatal diagnosis, and to explore possible genotype/phenotype correlations. Genomic DNA was isolated from unrelated affected males, and the entire alpha-Gal A coding region and flanking intronic sequences were analyzed by polymerase chain reaction (PCR) amplification and automated sequencing. Twenty new mutations were identified: M51K, D92N, D136H, F169S, C172F, L191Q, S247P, Q250X, P259R, G261D, T282N, R301P, W349X, T410K, 124delAT, 842delTAA, 1033delTC, 82insG, 893insG, and 903insG. In the remaining 20 unrelated Fabry families, 17 previously reported mutations were detected. These studies further define the heterogeneity of mutations in the alpha-Gal A gene causing the classic Fabry disease phenotype, and permit precise heterozygote detection and prenatal diagnosis.     

Detection of alpha-galactosidase a mutations causing Fabry disease by denaturing high performance liquid chromatography

Mutations in the alpha-galactosidase A (alpha-Gal A, GLA) gene cause Fabry disease, an X-linked recessive lysosomal storage disease. The majority of mutations are private, and confirmation of carrier status in females requires the definitive identification of a DNA mutation. In addition, knowledge of a family's mutation enables rapid and precise preimplantation and prenatal genetic testing. Here we report the development and use of DHPLC to rapidly and cost-effectively screen for alpha-Gal A mutations. Optimal DHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to the seven alpha-Gal A exons and their adjacent intronic/flanking sequences. At least five known mutations in each exon (45 in total) were screened by DHPLC to validate the method. Mutation detection was then performed in 14 affected males diagnosed by enzyme assay and 39 at-risk females, and the amplicons with abnormal DHPLC profiles were sequenced. In all affected males, and in 32 of the 39 at-risk females, four and 16 previously reported and 10 and 15 new mutations were identified, respectively. Sequencing all seven alpha-Gal A gene amplicons in the seven at-risk females who had normal DHPLC profiles excluded them as mutation carriers. Only one mutation (p.P362L) was not initially identified by its DHPLC profile, but in retrospect the profile was abnormal, emphasizing the need for experience in inspecting the profiles. In addition, this technique detected two new intronic polymorphisms, c.640-16A>G and c.1000-22C>T, with frequencies of 0.14 and 0.25 in both normal individuals and Fabry patients, respectively. This DHPLC method should improve the rapidity and cost-effectiveness of alpha-Gal A mutation identification in affected males and carrier females for Fabry disease.     

A previously undescribed frameshift deletion mutation of HFE (c.del277; G93fs) associated with hemochromatosis and iron overload in a C282Y heterozygote

A 62-year-old white man with a hemochromatosis phenotype was found to be heterozygous for the C282Y mutation of the HFE gene. The H63D and S65C mutations of HFE were not present. As most C282Y heterozygotes do not develop a hemochromatosis phenotype, the coding region of the patient's HFE gene was sequenced and a previously undescribed frameshift mutation was identified in exon 2 (c.del277; G93fs) that resulted in a premature stop-codon. There were no coding region mutations of the ferroportin gene (FPN1). We performed human leukocyte antigen (HLA) typing of the patient and his brother who was heterozygous for the C282Y HFE mutation unassociated with a hemochromatosis phenotype. They shared only C282Y and the HLA haplotype A*03, B*14; hence, the c.del277 mutation was linked to the HLA haplotype A*02, B*44 and therefore not on the same chromosome as the C282Y mutation. Thus, the present patient's only intact HFE protein is C282Y, and this may explain his hemochromatosis phenotype.     

Fabry disease: comparison of enzymatic, linkage, and mutation analysis for carrier detection in a family with a novel mutation (30delG).

Fabry disease (FD) is an X-linked recessive disorder caused by the deficient activity of the lysosomal enzyme alpha-galactosidase A (alpha-Gal A). Affected males are reliably diagnosed by demonstration of deficient alpha-Gal A activity in plasma or leukocytes. However, identification of female carriers is problematic due to Lyonization, requiring mutation identification and/or linkage studies for accurate carrier detection. Here, we describe a large Brazilian kindred with Fabry disease that permitted comparison of biochemical and molecular diagnostic techniques. Initially, the plasma alpha-Gal A activities were determined in at-risk affected males and potential female carriers; affected males were readily diagnosed, while the females had variable results. To detect carrier females, haplotype analysis using 10 polymorphic markers adjacent to the gene was performed. Subsequently, solid-phase direct sequencing of the alpha-Gal A gene demonstrated a novel single base deletion in exon 1 (30delG). Discrepancies were observed between the enzymatic and molecular diagnoses in two at-risk females. These findings emphasize the need for precise heterozygote diagnosis by mutation and/or haplotype analyses in all families with Fabry disease.     

Mutation analysis of the G4.5 gene in patients with isolated left ventricular noncompaction

Mutations in the gene G4.5, originally associated with Barth syndrome, have been reported to result in a wide spectrum of severe infantile X-linked cardiomyopathies. The purpose of this study was to investigate patients with isolated left ventricular noncompaction (LVNC) for disease-causing mutations in G4.5. In 27 patients including 10 families with isolated LVNC, mutation analysis of G4.5 was performed using single-strand DNA conformation polymorphism (SSCP) analysis and DNA sequencing. A novel splice acceptor site mutation of intron 8 of G4.5 was identified in a family with severe infantile X-linked LVNC without the usual findings of Barth syndrome. This mutation results in deletion of exon 9 from the mRNA, and is predicted to significantly disrupt the protein product. Genotype-phenotype correlation of G4.5 mutations in all 38 cases reported in the literature to date revealed that there was no correlation between location or type of mutation and either cardiac phenotype or disease severity. We suggest that males presenting with cardiomyopathy, particularly during infancy, even in the absence of the typical signs of Barth syndrome, should be evaluated for mutations in G4.5.     

The mutation p.D313Y is associated with organ manifestation in Fabry disease

Fabry disease (FD) is a multisystem lysosomal storage disorder caused by mutations in the GLA gene. The clinical significance of the mutation p.D313Y is still under debate. Retrospective chart analysis of clinical (neurological, cardiac, renal, and ophthalmological), genetic, and biochemical (lyso‐globotriaosylsphingosine, lyso‐Gb3; enzyme activity) data was performed in all our patients carrying the p.D313Y mutation. Fourteen patients from 5 families (10 female, 4 male; age range 10‐51) were included. Symptoms and organ manifestations compatible with FD could be identified in 10 patients. Cerebrovascular events occurred in 4 females. Seven patients reported pain or acroparaesthesia. Cornea verticillata was found in 1 patient, mild retinal vascular tortuosity in 5 patients. Lyso‐Gb3 was elevated in 2 females with cerebrovascular involvement. Classical cardiac, renal or skin manifestations could not be identified. The mutation p.D313Y in the GLA gene may lead to organ manifestations and elevation of the Fabry‐specific biomarker lyso‐Gb3. Neurological symptoms (stroke and pain) and ocular manifestations seem to be the leading findings. Annual routine visits are recommended for patients carrying the p.D313Y mutation. Enzyme replacement therapy might be considered in symptomatic patients.     

Identification of recurrent and novel mutations in the LDL receptor gene in Spanish patients with familial hypercholesterolemia. Mutations in brief no. 135. Online

We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(-18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706-10G->A and 2390-1G->A, and the other one has been reported recently, 313+1G->C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G->C; C95R and D679E; W(-18)X and E256K, and C281Y and 1706-10G->A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population.     

Identification of 9 novel IDS gene mutations in 19 unrelated Hunter syndrome (mucopolysaccharidosis Type II) patients. Mutations in brief no. 202. Online

Hunter syndrome is an X-linked lysosomal storage disorder caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The IDS deficiency can be caused by several different types of mutations in the IDS gene. We have performed a molecular and mutation analysis of a total 19 unrelated MPS II patients of different ethnic origin and identified 19 different IDS mutations, 9 of which were novel and unique. SSCP analysis followed by DNA sequencing revealed four novel missense mutations: S143F, associated with the 562C-->T polymorphism, C184W, D269V and Y348H. Two novel nonsense mutations were found: Y103X (433C-->A) and Y234X (826C-->G). In two patients two novel minor insertions (42linsA and 499insA) were identified. In one patient a complete IDS deletion was found, extending from locus DXS1185 to locus DXS466).     

Identification of 9 novel IDS gene mutations in 19 unrelated Hunter syndrome (Mucopolysaccharidosis type II) patients

Hunter syndrome is an X-linked lysosomal storage disorder caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The IDS deficiency can be caused by several different types of mutations in the IDS gene. We have performed a molecular and mutation analysis of a total of 19 unrelated MPS II patients of different ethnic origin and identified 19 different IDS mutations, 9 of which were novel and unique. SSCP analysis followed by DNA sequencing revealed four novel missense mutations: S143F, associated with the 562C→T polymorphism, C184W, D269V and Y348H. Two novel nonsense mutations were found: Y103X (433C→A) and Y234X (826C→G). In two patients two novel minor insertions (421insA and 499insA) were identified. In one patient a complete IDS deletion was found, extending from locus DXS1185 to locus DXS466. © 1998 Wiley-Liss, Inc.     

Different phenotypic expression in relatives with fabry disease caused by a W226X mutation

Two male relatives with Fabry disease presented striking differences in clinical symptoms and age of onset. The propositus had retarded statural growth and skeletal dysplasia while his nephew suffered mainly from aggravating acroparesthesia and celiac disease. Fabry disease is an X-linked inborn error of glycosphingolipid metabolism resulting from deficient activity of the lysosomal hydrolase alpha-galactosidase A (alpha-Gal A) enzyme. The alpha-Gal A gene is located at Xq22.1. Efforts to establish genotype-phenotype correlations have been limited because most patients have private mutations. In previous clinical studies performed in families with Fabry disease, marked differences in phenotype are described between affected relatives. This family also demonstrates the difficulty in predicting the clinical phenotype in patients and relatives with the same alpha-Gal A mutation. Furthermore, in the absence of a family history, the diagnosis may be easily missed.     

Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum

The dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional complex from pyrimethamine-sensitive (3D7) and drug-resistant (HB3 and 7G8) clones from Plasmodium falciparum was purified to homogeneity. A modified sequence of purification steps with a 10-formylfolate affinity column at its center, allows the isolation of the enzyme complex with a 10-fold higher yield than previously reported, irrespective of the pyrimethamine resistance of the parasites. Titration of the homogenous DHFR-TS complex with the inhibitor revealed a 500-fold lower affinity of the enzyme from clone 7G8 for the drug than found with the enzyme from clone 3D7. Direct comparison of the homogenous enzyme preparations on SDS-PAGE revealed no difference in the molecular mass of the DHFR-TS from the 3 clones, nor could a reproducible difference be detected in the peptide patterns obtained after digesting the DHFR-TS complex with various proteases. The amplification of segments from the DHFR-TS coding region of the 3 clones and 7 isolates of P. falciparum by polymerase chain reaction resulted in fragments of the predicted length without any size heterogeneity. The DNA sequence of the DHFR coding region from FCR-3, 3D7, HB3 and 7G8 differs in a total of 4 nucleotides. One point mutation changes amino acid residue 108 from threonine (FCR-3) or serine (3D7) to asparagine (HB3 and 7G8). The presence of asparagine-108 appears to be the molecular basis of pyrimethamine resistance of HB3 and 7G8. The degree of resistance is associated with a point mutation affecting the codon for amino acid 51 in 7G8.     

低密度脂蛋白受体相关蛋白5基因的基因组结构

目的 确定2低密度脂蛋白受体相关蛋白5（low density lipoprotein receptor related protein,5 LRP5）的基因组结构。方法 以LRP5基因的cDNA序列为线索，采用计算机杂交方法首先通过对比分析该基因的cDNA序列和基因组序列，初步确定LRP5基因的基因组结构，按分析得到的基因组结构设计引物，扩增并测定外显子序列和外显子内含子接头序列，确定该基因的基因组结构。结果 LRP5基因的基因组DNA全长为131.6kb，含4有23个外显子和22个内含子；在编码序列中检测到3个单核苷酸多态，即位于第2外显子的A459G，位于第10外显子的C2220T和位于第21外显子的G4416C；LRP5基因含有4个已知的短串联重复序列，即D11S1917，D11S4087，D11S1337和D11S4178，它们分别位于该基因的5'端和第1，4，13内含子内。结论 LRP5基因的基因组结构的确定，为分析该基因突变和功能研究奠定了基础。     

A previously undescribed nonsense mutation of the HFE gene

A patient with clinically manifest hereditary hemochromatosis was found to be heterozygous for the c.845 A-->G (C282Y) mutation. As simple heterozygotes for this mutation do not develop the hemochromatosis phenotype, the coding region of the patient's HFE gene was sequenced and a previously undescribed nonsense mutation was identified at c.211 C-->T (R74X). The patient's brother who also had the hemochromatosis phenotype shared his HFE genotype. To determine how common such mutations might be, the coding and 5' region of the HFE genes of 11 subjects who had been found in a large population survey to be heterozygous for the C282Y mutation and had elevated ferritin levels were sequenced. No mutations were found. Sequencing of the HFE gene also revealed two polymorphisms that had not previously been noted, -467 C-->G and -970 T-->G. Neither of these mutations appear to cause an abnormality in iron metabolism.     

一个先天性白内障家系缝隙连接蛋白基因新突变

目的 鉴定一个中国常染色体显性遗传先天性白内障(autosomal dominant congenital cataract,ADCC)家系其致病与缝隙连接蛋白a3/a8(gap junction protein alpha3/alpha8,GJA3/GJA8)基因突变的关系.方法 对一个ADCC家系5名家系成员和100名正常健康人进行全面眼科检查.抽取外周血5 mL并提取基因组DNA.采用聚合酶链反应扩增GJA3/CJA8编码外显子及其侧翼内含子序列,纯化的PCR产物通过直接测序以筛查致病突变.结果 通过双向序列分析,发现GJA8基因第2外显子的第138位发生碱基G→A转换(c.138G》A,GGG→GGA),产生同义突变(G46G);第139个碱基因G→T颠换发生错义突变(c.139 G》T,GAT→TAT)导致第47位编码天门冬氨酸密码子突变为酪氨酸(D47Y),家系中非患者和100名对照者基因组序列均无此改变,而且生物信息检索显示GJAB基因第47位编码的天门冬氨酸具有高度的物种保守性.家系3例患者CJA3基因突变筛查未见任何碱基改变.结论 在一个中国人ADCC家系中发现GJAB基因新致病突变(D47Y).     

Neanthes japonica (Iznka) fibrinolytic enzyme reduced cerebral infarction, cerebral edema and increased antioxidation in rat models of focal cerebral ischemia

X-linked retinitis pigmentosa (XLRP) is the most severe type of retinitis pigmentosa (RP), with patients consistently showing early onset and rapid deterioration. Obtaining a genetic diagnosis for a family with XLRP is important for counseling purposes. In this study, we aimed to identify disease-causing mutations in two unrelated XLRP families. Genetic analysis was performed on two unrelated XLRP families. Genomic DNA was extracted from peripheral blood or amniotic fluid samples. The coding regions and intron/exon boundaries of the Retinitis Pigmentosa GTPase Regulator (RPGR) and RP2 genes were amplified by PCR and then sequenced directly. A clinically unaffected pregnant female and the four month old fetus were found to have a hemizygous 2 base pair deletion (g.ORF15+484_485delAA) in the exon ORF15 of RPGR gene. In another XLRP family, a nonsense mutation (g.ORF15+810G>T) was identified. Neither mutation has been reported previously. Both are predicted to cause premature termination of the protein. In conclusion, we identified a micro-deletion through prenatal genetic diagnosis and another novel nonsense mutation in RPGR-ORF15. Identifying a disease-causing mutation facilitated early diagnosis and genetic counseling for the patients. Discovery of novel mutations also broadens knowledge of XLRP and the spectrum of its pathogenic genotypes.     

Remarkable variability in renal disease in a large Slovenian family with Fabry disease

Following the diagnosis of Fabry disease in a 45-year-old male, in 31 family members alpha-galactosidase A (alpha-Gal) activity in leucocytes was measured and mutation analysis of the alpha-Gal gene was performed. In the proband, the unique mutation A10523G/N272S in exon 6 was found, which was subsequently detected in seven males (of which one twin) and 10 female subjects. All males showed decreased to absent alpha-Gal A activity in leucocytes, but three out of 10 female subjects had alpha-Gal A activities within normal range. Although all male patients had symptoms of classical Fabry disease, such as acroparesthesias, hypohydrosis and heat-intolerance, there was considerable variability in organ involvement, especially in deterioration of renal function. Detailed studies of large families with Fabry disease may give insight into factors that influence the phenotype of this disorder.