Plasminogen with type-I mutation is polymorphic in the Japanese population

Summary A functionally inactive plasminogen (PLG) variant designated as PLG M5 is polymorphic in the Japanese population and has a feature common to PLG with type-I mutation that has a codon 601 missense mutation in exon 15 (GCT for AlaACT for Thr). This study was conducted to clarify whether the type-I mutation of PLG is present in PLG M5 and polymorphic in the Japanese population. Direct sequencing of the amplified DNA from the PLG gene in a heterozygote for PLG M5 revealed that the sequence of the exon 15 in the gene for PLG M5 is identical with that in the PLG gene with type-I mutation. In addition, the amplified DNA from the PLG gene in 12 heterozyotes for PLG M5 reacted with the probe for the type-I mutation in dot blot hybridization with an allele-specific oligonucleotide probe. The heterozygote for PLG with type-I mutation was found in 2.2% of 360 unrelated healthy subjects. These data indicate that the type-I mutation of PLG is present in PLG M5 and polymorphic in the Japanese population. The data also suggest that the PLG M5 is identical with PLG Tochigi and Kagoshima.     

Mucopolysaccharidosis I: a comparative study of haplotypes Eco47III-NspI sites frequencies in patients and healthy subjects of Mexican population

BACKGROUND: Mucopolysaccharidosis I (MPS-I) is an autosomal recessive disorder, which is caused by mutations in the IDUA gene. It induces the deficiency of glycosidase alpha-L-duronidase. The enzyme that is required for the degradation of heparan and dermatan sulfate. This disorder expresses a wide range of clinical symptoms (severe mental retardation, skeletal deformations, hepatosplenomegaly, corneal clouding and mild visceral organ involvement). In the present paper, we report the frequencies of haplotypes of the Eco47III-NspI sites, in the IDUA gene, in Mexican healthy and in MPS-I individuals. METHODS: Eco47III and NspI intragenic polymorphisms in IDUA gene were studied in 262 (524 chromosomes) Mexican healthy subjects and in 53 (106 chromosomes) MPS-I patients. RESULTS: The genotypes for IDUA Eco47III and NspI sites in Mexicans were in agreement with Hardy-Weinberg expectations. Allele frequency distributions for individual sites differed (P < 0.05) in both groups. Haplotype Eco47III-NspI frequencies of Mexican MPS-I patients also differed from those of the normal Mexican population. The data provide evidence of linkage disequilibrium, since the MPS-I group constitutes a subset of the Mexican control population. The disequilibrium in Mexican MPS-I patients was defined by an increase in the haplotype A1B2, and deficiency in A2B1, with respect to normal population (P < 0.05). CONCLUSIONS: Our results support that these polymorphisms can be associated to mutations in IDUA gene, which leads to MPS-I in Mexican patients. On the other hand, these polymorphisms can be used to identify heterozygosity when they are informative.     

Mucopolysaccharidosis IVA: characterization of a common mutation found in Finnish patients with attenuated phenotype

Mucopolysaccharidosis IVA (MPS IVA) is caused by the deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase encoded by the GALNS gene on chromosome 16. We describe, in detail, the clinical phenotype of five patients from three unrelated Finnish families and have characterized the disease-causing mutations in GALNS. Genotypes of the patients are D60N/A291T, D60N/W230X, and D60N/1374delT. Mutation 1374delT introduces premature termination of GALNS. Cells over-expressing the novel mutation W230X and A291T had no residual GALNS activity, whereas D60N gave 12.2% residual activity compared with the wild type. Co-transfection of D60N/A291T and D60N/W230X showed 5.5% and 6.7% of wild type activity, respectively. The precursor proteins of D60N and A291T were observed at 55 kDa and 57 kDa, respectively, whereas there was no detectable band in cells over-expressing W230X. At 55 degrees C, the mutant protein showed lower thermostability than the wild type protein at pH 3.8 and 7.0. The tertiary structural model of the GALNS protein revealed that aspartic acid at position 60 is located on the surface of the molecule, away from the active site. This makes it unlikely that the enzymatic function of the protein with D60N is severely impaired. On the other hand, A291 and W230 are localized near the active site. The molecular characteristics of the D60N mutation explain the attenuated clinical phenotype of the patients.     

Mucopolysaccharidosis IVA: four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency.

We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations. V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resulted in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Mucopolysaccharidosis type IVA: common double deletion in the N-acetylgalactosamine-6-sulfatase gene (GALNS)

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). We found two separate deletions of nearly 8.0 and 6.0 kb in the GALNS gene, including some exons. There are Alu repetitive elements near the breakpoints of the 8.0-kb deletion, and this deletion resulted from an Alu—Alu recombination. The other 6.0-kb deletion involved illegitimate recombinational events between incomplete short direct repeats of 8 bp at deletion breakpoints. The same rearrangement has been observed in a heteroallelic state in four unrelated patients. This is the first documentation of a common double deletion a gene that is not a member of a gene cluster.     

Mucopolysaccharidosis IVA: Identification of a Common Missense Mutation I113F in the N-Acetylgalactosamine-6-Sulfate Sulfatase Gene

Mucopolysaccharidosis IVA is an autosomal recessive lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase. The recent isolation and characterization of cDNA and genomic sequences encoding GALNS has facilitated identification of the molecular lesions that cause MPS IVA. We identified a common missense mutation among Caucasian MPS IVA patients. The mutation was originally detected by SSCP, and successive sequencing revealed an A→T transversion at nt 393. This substitution altered the isoleucine at position 113 to phenylalanine (I113F) in the 622 amino acid GALNS protein and was associated with a severe phenotype in a homozygote. Compound heterozygotes with one I113F-allele mutation have a wide range of clinical phenotypes. Transfection experiments in GALNS-deficient fibroblasts revealed that the mutation drastically reduces the enzyme activity of GALNS. Allele-specific oligonucleotide or SSCP analysis indicated that this mutation accounted for 22.5% (9/40) of unrelated MPS IVA chromosomes from 23 Caucasian patients, including 6 consanguineous cases. Of interest, the I1e 113→Phe substitution occurred in only Caucasian MPS IVA patients and in none of the GALNS alleles of 20 Japanese patients. These findings identify a frequent missense mutation among MPS IVA patients of Caucasian ancestry, that results in severe MPS IVA when homoallelic, and will facilitate molecular diagnosis of most such patients and identification of heterozygous carriers. In addition to this common mutation, 10 different point mutations and 2 small deletions were detected, suggesting allelic heterogeneity in GALNS gene.     

Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear

Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB(-α-γ)) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649?kb around S(4)-RNase and 378?kb around S(2)-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB(4-u1-u4, 4-d1-d2) and PpSFBB(2-u1-u5,) (2-d1-d5), respectively) were found, but PpSFBB(4-α-γ) and PpSFBB(2-γ) were absent. The PpSFBB(4) genes showed 66.2-93.1% amino acid identity with the PpSFBB(2) genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3-94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.     

MspI polymorphic site in intron 22 of the factor VIII gene in the Japanese population

Summary A novel MspI DNA polymorphic site has been found in intron 22 of the human factor VIII gene. This site is informative almost exclusively in the Japanese population (heterozygosity 0.45) and will be of considerable importance in carrier detection and prenatal diagnosis of hemophilia A in this population.     

Enzyme Replacement in a Human Model of Mucopolysaccharidosis IVA In Vitro and Its Biodistribution in the Cartilage of Wild Type Mice

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K_uptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.     

Cystic fibrosis gene mutations and linked RFLPs in the Slovenian population.

The authors used polymerase chain reaction to analyse 56 Slovenian cystic fibrosis (CF) chromosomes for the presence of delta F508 and eight other most frequent mutations located in exons 7,11 and 20 (R347P, R334W, G551D, R553X, S549RA, S549RT, S549I and S1255X) of the CF gene. We also determined the frequency of haplotypes associated with CF for six linked RFLP markers (MetD/TaqI, MetH/TaqI, XV-2c/TaqI, KM-19/PstI, MP6d9/MspI and J3.11/MspI) in 27 Slovenian CF families. delta F508 mutation was present in 55.4 percent of the CF chromosomes. No case of the other mutations were detected in the sample of tested CF chromosomes. A very high degree of association (0.88) has been found between DNA marker MetH and CF (as measured by the Yule's association coefficient) in our population. Using the RFLP markers XV-2c and KM-19, we found that 85% of delta F508 mutated chromosomes have a single 1 2 (B) haplotype, and that this haplotype is present on only 15.4 percent of CF chromosomes without this deletion.     

Insights into Iberian population origins through the construction of highly informative Y-chromosome haplotypes using biallelic markers,STRs, and the MSY1 minisatellite

To investigate the diversity of Y chromosomes in the Iberian Peninsula and the North African population of Maghreb, we constructed superhaplotypes on the basis of 10 biallelic markers, 7 microsatellites, and 1 minisatellite located in the nonrecombining portion of the human Y chromosome. The analysis of extremely high MSY1 variability was performed by reducing the MVR-codes to modular structures. Y-STRs and MSY1 data provide information about the relationship between closely related populations such as those of Iberia. Analysis of biallelic markers allowed us to identify 7 of 12 haplogroups defined by those polymorphisms. The haplogroup background showed clear differences between Iberian populations and the North African one. The use of differently mutating Y-chromosome markers allowed us to infer different population events at different time scales: the Paleolithic background of the Iberian Peninsula, the Neolithic fingerprint on Y-chromosome lineages, and the Iron Age influence in the populations of Iberia. Implications of our results for the highly debated origin of Basques are also discussed.     

CFTR haplotypes in northern Iranian population

Background

Cystic fibrosis (CF) is a multiorganic autosomal recessive disorder, caused by mutation in cystic fibrosis transmembrane conductance regulator (CFTR). CF is highly heterogeneous in Iranian population and molecular diagnosis based on direct identification of mutations is not completely efficient. The use of polymorphic intragenic markers not only can facilitate phenotype prediction in prenatal diagnosis by gene tracking, but also can lead to the demonstration of possible associations between haplotypes and specific mutations.

Methods

60 CF patients and 53 fertile normal subjects originating from North of Iran were analyzed for F508del mutation and c.1210-12T(5_9), c.1408A>G and c.744-33GATT(6_8) polymorphisms.

Results

c.1210-12T[7] is the most prevalent allele in normal individuals and CF non-F508del patients with 87.7%and 86.7% frequencies respectively. c.1408A>G survey showed that frequency of allele G and A is nearly equal in both non-F508del CF patients and normal individuals. c.744-33GATT(6_8) study showed that 7 repeat is the most prevalent allele in normal individuals and non-F508del CF patients with 80.2% and 82.1% frequencies respectively. The [c.1408A; c.1210-12T[9]; c.744-33GATT[6]] haplotype was only associated with mutant alleles including F508del.

Conclusions

The allelic distribution and heterozygosity results suggest that c.1408A>G, c.1210-12T(5_9) and c.744-33GATT(6_8) can contribute to carrier detection and prenatal diagnosis of CF in Iranian families with previous history of the disease.     

Two distinct subtypes of theHLA-DRw12 haplotypes in the Japanese population detected by nucleotide sequence analysis and oligonucleotide genotyping

We determined the DNA sequence of the enzymatically amplified second exon of theDRB1 gene of theDRw12 haplotypes derived from three Japanese donors and found two distinct subtypes of theDRw12 haplotype. The two subtypes, designatedDRw12a andDrw12b, had single-base substitutions that predicted one amino acid change at residue number 67. The sequence of theDrw12a andDRw12b subtypes differed from those of the otherDR haplotypes, but in the first hypervariable region of theDRB1 gene the sequences were identical to those of theDRw8(Dw8.1) andDRw8(Dw8.3) haplotypes. TheDRw12a andDRw12b subtypes were detected in a wide range of Japanse donors by genotyping with sequence-specific oligonucleotide probes synthesized according to the DNA sequence of the two subtypes. Results of this study demonstrated that theDRw12 haplotypes in the Japanese population are genetically diverse, as many otherDR haplotypes are. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M27509, M27510, M27511.     

Analysis of mitochondrial DNA haplotypes in yakut population

To study the mitochondrial gene pool structure in Yakuts, polymorphism of mtDNA hypervariable segment I (16,024-16,390) was analyzed in 191 people sampled from the indigenous population of the Sakha Republic. In total, 67 haplotypes of 14 haplogroups were detected. Most (91.6%) haplotypes belonged to haplogroups A, B, C, D, F, G, M*, and Y, which are specific for East Eurasian ethnic groups; 8.4% haplotypes represented Caucasian haplogroups H, HV1, J, T, U, and W. A high frequency of mtDNA types belonging to Asian supercluster M was peculiar for Yakuts: mtDNA types belonging to haplogroup C, D, or G and undifferentiated mtDNA types of haplogroup M (M*) accounted for 81% of all haplotypes. The highest diversity was observed for haplogroups C and D, which comprised respectively 22 (44%) and 18 (30%) haplotypes. Yakuts showed the lowest genetic diversity (H = 0.964) among all Turkic ethnic groups. Phylogenetic analysis testified to a common genetic substrate of Yakuts, Mongols, and Central Asian (Kazakh, Kyrgyz, Uigur) populations. Yakuts proved to share 21 (55.5%) mtDNA haplogroups with the Central Asian ethnic groups and Mongols. Comparisons with modern paleo-Asian populations (Chukcha, Itelmen, Koryaks) revealed three (8.9%) haplotypes common for Yakuts and Koryaks. The results of mtDNA analysis disagree with the hypothesis of an appreciable paleo-Asian contribution to the modern Yakut gene pool.