Tight linkage of pyruvate kinase (PKLR) and glucocerebrosidase (GBA) genes

Two polymorphisms, one in the liver-type pyruvate kinase gene (PKLR) and one in the glucocerebrosidase gene (GBA), both of which are on band q21 of chromosome 1, were found to be tightly linked. Each of three Gaucher disease mutations in 112 chromosomes studied was associated with a unique haplotype. With a conservative assumption about the length of time that the Gaucher disease mutation has been present in the Jewish population, we deduce that the genetic distance between these two loci is probably under 0.2 centimorgans. Four haplotypes are produced by these polymorphic loci, but two of these are relatively uncommon because the polymorphic sites are in linkage disequilibrium. Nonetheless these markers are potentially useful in the prenatal diagnosis of pyruvate kinase deficiency in families who have at least one affected child and may also be helpful in heterozygote detection in families with Gaucher disease where a specific mutation producing the disease in unknown.     

Extensive restriction site polymorphism at the human phenylalanine hydroxylase locus and application in prenatal diagnosis of phenylketonuria.

A total of 10 restriction site polymorphisms have been identified at the human phenylalanine hydroxylase locus using a full-length human phenylalanine hydroxylase cDNA clone as a hybridization probe to analyze human genomic DNA. These polymorphic patterns segregate in a Mendelian fashion and concordantly with the disease state in various PKU kindreds. The frequencies of the restriction site polymorphisms at the human phenylalanine hydroxylase locus among Caucasians are such that the observed heterozygosity in the population is 87.5%. Thus, most families with a history of classical phenylketonuria can take advantage of the genetic analysis for prenatal diagnosis and carrier detection of the hereditary disorder.     

Phenylketonuria: distribution of DNA diagnostic patterns in German families

Summary The distribution of DNA haplotype constellations within the phenylalanine hydroxylase (PAH) gene was investigated in 44 German families affected with phenylketonuria (PKU). The haplotype frequencies differed significantly from those observed in a Danish population. Furthermore, ten haplotypes were identified in addition to the 12 previously described. In one of ten PKU alleles linked to haplotype 3, the G to A transition at the 5 splice donor site of intron 12 could not be confirmed with the use of synthetic DNA probes. According to these data, which are still limited, carrier testing and prenatal diagnosis should be possible in 70% of individuals at risk in the German population.     

Linkage of the gene for an X-linked mental retardation disorder to a hypervariable (AGAT)n repeat motif within the human hypoxanthine phosphoribosyltransferase (HPRT) locus (Xq26).

We recently reported a new X-linked mental retardation (XLMR) disorder in a four-generation family of Dutch descent. Features included Dandy-Walker malformation, basal ganglia disease, and seizures. Twenty-six family members, including two living affected males and two obligate carriers, were available for study. No evidence of linkage was observed between the disease locus and RFLPs from several X-chromosome regions, including Xp21-p22 (13 markers), proximal Xq (four markers), and Xq28 (three markers). However, a new hypervariable short tandem repeat (STR) within the HPRT gene at Xq26 showed positive linkage to the disease locus, with a maximum lod score of 2.19 at a recombination fraction of 0. A second hypervariable marker in Xq26, the dinucleotide repeat XL90A3 (DXS425), showed a lod score of .84 at a recombination fraction of .11. Both the HPRT and DXS425 markers were typed in 40 CEPH families, and subsequent multipoint linkage analysis showed the following order: Xcen-DXS425-(HPRT,XLMR)-F9-qter. HPRT and these flanking markers are therefore useful for carrier detection and prenatal diagnosis in this family. This study illustrates that hypervariable STRs will be powerful tools for linkage analysis and genetic diagnosis, particularly when relatively small families are involved.     

Compound heterozygosity in nonphenylketonuria hyperphenylalanemia: the contribution of mutations for classical phenylketonuria

Hyperphenylalaninemia (HPA) results from defective hydroxylation of phenylalanine in the liver, in most cases because of defective phenylalanine hydroxylase. HPA is highly variable, ranging from moderate elevation of plasma phenylalanine with no clinical consequences to a severe disease, classical phenylketonuria (PKU). Non-PKU HPA was found in excess of PKU in Israel, while the opposite is true in Europe. To study the genetic basis of non-PKU HPA, we performed haplotype analysis at the phenylalanine hydroxylase locus in 27 families with non-PKU HPA. All individuals with this condition were compound heterozygotes. In six of these families, in which both PKU and non-PKU HPA were segregating, haplotype analysis showed that non-PKU HPA resulted from compound heterozygosity for a PKU mutation and a second mutation, with milder effect, which is probably expressed only when it interacts with the severe mutation. The involvement of PKU mutations in non-PKU HPA was further demonstrated in Jewish Yemenite families with non-PKU HPA, in which the individuals with this condition were carriers of the single PKU allele which exists in this community. In addition, two previously known PKU point mutations (R261Q and R408W) were found in individuals with non-PKU HPA. These mutations are associated, in our population, with the same haplotypes as those with which it is associated in Europe. Based on the above-mentioned genetic model for non-PKU HPA, successful prenatal diagnosis of this condition was performed in one family.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assignment of X-linked hydrocephalus to Xq28 by linkage analysis

X-linked recessive hydrocephalus (HSAS) occurs at a frequency of approximately 1 per 30,000 male births and consists of hydrocephalus, stenosis of the aqueduct of Sylvius, mental retardation, spastic paraparesis, and clasped thumbs. Prenatal diagnosis of affected males by ultrasonographic detection of hydrocephalus is unreliable because hydrocephalus may be absent antenatally. Furthermore, carrier detection in females is not possible because they are asymptomatic. Using four families segregating HSAS, we performed linkage analysis with a panel of X-linked probes that detect restriction fragment length polymorphisms. We report here that HSAS, in all tested families, is closely linked to marker loci mapping in Xq28 (DXS52, lod = 6.52 at theta of 0.03; F8, lod = 4.32 at theta of 0.00; DXS15, lod = 3.40 at theta of 0.00). These data assign HSAS to the gene-dense chromosomal band Xq28 and allow for both prenatal diagnosis and carrier detection by linkage analysis.     

Two highly polymorphic CA repeats in the Menkes gene (ATP7A)

Two highly polymorphic CA repeats have been identified in the Menkes gene (ATP7A). These repeats should be useful for prenatal diagnosis and carrier detection in families with Menkes disease and X-linked cutis laxa. The observed heterozygosity for these two repeats was 0.778 and 0.60 in Centre d'Etude du Polymorphisme Humaine (CEPH) families.     

Close linkage of probe p212 (DXS178) to X-linked agammaglobulinemia

Summary Segregation analysis was performed in three families affected in X-linked agammaglobulinemia (XLA) with five polymorphic DNA probes linked to the disease locus. In agreement with previous studies, no recombination was observed with either pXG12 (DXS94) or S21 (DXS17). Segregation analysis was also performed with a marker, p212 (DXS178), which has been shown to be closely linked to pXG12 in normal families. No cross-over with XLA was observed in these three families and in five additional families previously analyzed with DXS17 and DXS94 (z = 5.92 at = 0). These data provide evidence against genetic heterogeneity in XLA and indicate the value of probe p212 for carrier detection and prenatal diagnosis of XLA. We were able to estimate the carrier status of six females (out of six) in the three previously unreported families.     

Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms.

To improve carrier detection and prenatal diagnosis for Duchenne and Becker muscular dystrophy families, we determined allele frequencies and measures of variation for four (dC-dA)n.(dG-dT)n loci identified within a deletion-prone region of the human dystrophin gene. The loci are highly polymorphic, with predicted heterozygosities of 71.6%-93.3%. Direct DNA sequence analysis of the (dC-dA)n.(dG-dT)n locus in intron 49 revealed an additional length polymorphism which varies by single-basepair increments, is adjacent to the dinucleotide repeat block, and enhances the polymorphic content of this marker. The four (dC-dA)n.(dG-dT)n loci are each easily amplified by PCR in two diplex reactions. The variability of allele lengths at these loci makes them ideal for carrier detection and prenatal diagnosis, often providing diagnostic information when RFLP analysis is uninformative. These markers have aided in identification of deletion mutations, exclusion of maternal cell contamination of chorionic villus samples, confirmation of paternity, and mapping of gene recombinations. The allele identification of these loci can be performed either with a radiolabel or with an automated, nonradioactive, fluorescent gel detection system.     

Missense mutations prevalent in Orientals with phenylketonuria: molecular characterization and clinical implications

Two missense mutations in the phenylalanine hydroxylase (PAH) genes of Orientals with phenylketonuria (PKU) have been identified. A G-to-A transition in exon 7 of the gene results in the substitution of Gln243 for Arg243 (R243Q) and accounts for 18% of all PKU chromosomes among Chinese. An A-to-G transition in exon 6 of the gene results in the substitution of Cys204 for Tyr204 (Y204C) and identifies about 13 and 5% of all PKU chromosomes in the Chinese and Japanese populations, respectively. The R243Q construct produced less than 10% of normal PAH activity in in vitro expression analysis in a eukaryotic cell system, and patients homozygous for this substitution exhibit a severe clinical phenotype. These results are consistent with previous findings in this expression system. The Y204C construct, however, produced near normal levels of PAH enzyme activity and immunoreactivity in this in vitro expression system. Because this substitution is present only on PKU chromosomes, it is a valuable marker for identifying the corresponding mutant allele for carrier screening of PKU. With the characterization of these two substitutions, about 60% of PKU alleles in China can now be identified. The continuing search for additional PKU mutations will permit effective carrier screening and prenatal gene diagnosis of PKU in East Asia.     

Phenylketonuria in Iranian population: a study in institutions for mentally retarded in Isfahan

Phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene (12q22-q24) resulting in a primary deficiency of the PAH enzyme activity, intolerance to the dietary intake of phenylalanine (Phe) and production of the phenylketonuria (PKU) disease. To date there have been no reports on the molecular analysis of PKU in Iranian population. In this study, the states of the PKU disease in terms of prevalence and mutation spectrum among patients reside in the institutions for mentally retarded in Isfahan was investigated. In the first step, 611 out of 1541 patients with PKU phenotype or severe mental retardation were screened for the PKU disease using the Guthrie bacterial inhibition assay (GBIA) followed by HPLC. Among the patients screened 34 (5.56%) were found positive with abnormal serum Phe of above 7mg/dl. In the next step, the presence of 18 common mutations of the PAH gene in 26 of the patients with classical PKU (serum Phe above 20mg/dl) was investigated, using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Of the 52 independent mutant alleles that were analyzed, 34 (65.38%) were genotyped showing 8 mutations as follows: R252W (15.38%), Q232Q (13.46%), R261Q (7.69%), delL364 (7.69%), IVS10-11g>a (5.77%), L333F (5.77%), V245V (5.77%) and S67P (3.85%). The results from this study may serve as a reference to analyze the PKU mutations in other part of Iran, and to establish diagnostic tests for carrier detection and prenatal diagnosis of the PKU disease in Iranian population.     

Polymorphic DNA haplotypes at the phenylalanine hydroxylase locus and their relation to phenotype in Swedish phenylketonuria families

Summary The genetic heterogeneity at the phenylalanine hydroxylase (PAH) locus was studied in 88 families including 93 of the 105 children with phenylketonuria (PKU) or hyperphenylalaninemia (HPA) detected through the Swedish neonatal screening program from 1966 to the end of 1986. Haplotypes based on eight restriction fragment length polymorphisms (RFLPs) at the PAH locus could be constructed for 132 normal and 136 mutant alleles. The normal alleles were of 27 different RFLP haplotypes, 9 of which have not been described previously, but there was a dominance of a few haplotypes common to many European populations. The distribution of mutant alleles was significantly different from that in neighboring countries, even though over 90% of all mutant alleles were confined to six RFLP haplotypes, also prevalent in other European populations. Allele-specific oligonucleotide hybridization analysis for the Arg⁴⁰⁸ to Trp⁴⁰⁸ mutation and for the G to A splicing mutation in intron 12 showed exceptions to the previously reported linkage of these mutations to mutant haplotypes 2 and 3, respectively. Correlation of mutant alleles with clinical phenotypes pointed to the presence of at least two different mutations associated with each of six haplotypes. We argue that PKU/HPA in the Swedish population may be caused by at least 13 different mutations in addition to the 4 already identified. The theoretical informativity of RFLP analysis in heterozygote detection and prenatal diagnosis in PKU/HPA families was estimated at approximately 85%. Carrier detection could, in effect, be accomplished for 88% of the 56 healthy siblings in the families studied.     

Identification and application of additional restriction fragment length polymorphisms at the human ornithine transcarbamylase locus.

Two additional restriction fragment length polymorphisms (RFLPs) have been identified at the human ornithine transcarbamylase (OTC) locus. Approximately 11% of women are heterozygous for an RFLP characterized by polymorphic bands at 3.7 and 3.6 kilobasepairs (kbp) observed after DNA digestion with TaqI. Twenty-nine percent of women are heterozygous for an RFLP characterized by polymorphic bands at 18.0 and 5.2 kbp observed after digestion with BamHI. Thus, in combination with the previously reported RFLPs identified using MspI, the X chromosomes in approximately 80% of women at risk for having a son with OTC deficiency are distinguishable by RFLPs at the OTC locus. Furthermore, we show that these RFLPs will be useful in families for prenatal diagnosis of OTC deficiency, carrier detection, and carrier exclusion.     

A dimorphic 4-bp repeat in the cystic fibrosis gene is in absolute linkage disequilibrium with the delta F508 mutation: implications for prenatal diagnosis and mutation origin.

The gene causing cystic fibrosis (CF) has been recently cloned, and the major mutation (delta F508) accounting for approximately 70% of CF chromosomes has been uncovered. We have identified at the 3' end of intron 6 in the CF gene a 4-bp tandem repeat (GATT) that exhibits interesting features. First, PCR screening of 103 normal individuals revealed that the repeat exists only in two polymorphic allelic forms, either as a hexamer or a heptamer. These two alleles are in Hardy-Weinberg equilibrium and predict a heterozygote frequency of 41% (p[seven repeats] = .71; q [six repeats] = .29). Second, the allele with six repeats was found linked to delta F508 on all 76 CF chromosomes investigated, demonstrating strong linkage disequilibrium and suggesting that delta F508 had originated on the gene bearing six repeats. Third, when the repeat alleles are linked to the DNA markers XV2c and KM19, extended haplotypes are generated. These new haplotypes become informative in situations in which prenatal diagnosis cannot be performed solely with XV2c and KM19. Since this repeat marker is located in the CF gene and would be very less likely to recombine with the gene, it can serve as a valuable DNA marker for haplotype analysis. A possible crossover, however, was identified between XV2c and KM19, transferring delta F508 to a different haplotype.     

Study of restriction fragment length polymorphisms at the human phenylalamine hydroxylase locus and evaluation of its potential application in prenatal diagnosis of phenylketonuria in Chinese

Summary Using a human phenylalanine hydroxylase cDNA probe, the restriction fragment length polymorphisms at the human phenylalanine hydroxylase locus have been determined with the restriction enzymes BglII, PvuII, EcoRI+BamHI, MspI, XmnI, HindIII and EcoRV. The frequency of the observed heterozygosity of the restriction site polymorphisms at this locus in a Chinese population is approximately 54%, which is significantly lower than that in Caucasians. No DNA rearrangement or deletion of the phenylalanine hydroxylase locus was detected among mutant phenylalanine hydroxylase genes in seven Chinese classical phenylketonuria (PKU) families. Haplotype analysis of these seven families revealed that the mutant alleles belonged to five different haplotypes, i.e. haplotype 4, 11 and three unreported haplotypes. The majority of normal and mutant phenylalanine hydroxylase genes are confined to hyplotype 4. These results indicate that approximately 42% of Chinese PKU families are informative for prenatal diagnosis of PKU when eight restriction sites linked to the phenylalanine hydroxylase locus are examined.     

Ethnic polymorphism of DXS52 (St 14) locus linked to coagulation factor VIII gene in Argentina

Sixty-five individuals belonging to 16 argentinian families of hemophilia A were studied using the St 14 probe (DXS52 locus). This probe is widely used for carrier detection and prenatal diagnosis, despite the risk of recombination between the factor VIII gene and the DXS52 locus, because of its high informativity. The families are divided in two groups: one group constituted only of metis of Indians according to interview and morphotype and a second group of caucasoids (Spanish essentially and Italian). In this study we have shown some ethnic variations of the TaqI RFLPs in the DXS52 locus. In the allelic system I, (which alleles are numbered from 1 to 8) we have noted an over representation of the larger alleles (2 and 3) and of the allele 8 in both Argentinian groups when compared to the caucasian population already studied in our laboratory. The additional polymorphic TaqI site giving the beta band in the system II (alpha and beta bands) is found more frequently in the Argentinian families than in Caucasians. Some other additional polymorphic sites have been found in generally constant bands giving additional allelic systems, in metis families.     

Twenty novel mutations in the alpha-galactosidase A gene causing Fabry disease

BACKGROUND: 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 30 unrelated families was determined to provide precise heterozygote detection, prenatal diagnosis, and define genotype-phenotype correlations. MATERIALS AND METHODS: Genomic DNA was isolated from affected males and/or carrier females from 30 unrelated families with Fabry disease. The entire alpha-Gal A coding region and flanking intronic sequences were analyzed by PCR amplification and automated sequencing. RESULTS: Twenty new mutations were identified, each in a single family: C142R, G183D, S235C, W236L, D244H, P259L, M267I, I289F, Q321E, C378Y, C52X, W277X, IVS4(+4), IVS6(+2), IVS6(-1), 35del13, 256del1, 892ins1, 1176del4, and 1188del1. In the remaining 10 unrelated Fabry families, 9 previously reported mutations were detected: M42V, R112C, S148R, D165V, N215S (in 2 families), Q99X, C142X, R227X, and 1072del3. Haplotype analysis using markers closely flanking the alpha-Gal A gene indicated that the two patients with the N215S lesion were unrelated. The IVS4(+4) mutation was a rare intronic splice site mutation that causes Fabry disease. CONCLUSIONS: These studies further define the heterogeneity of mutations in the alpha-Gal A gene causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis, and help delineate phenotype-genotype correlations in this disease. 相似文献   

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)₅ and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.     

Linkage disequilibrium and linkage analysis of the glucose-6-phosphatase gene

Recent studies have indicated that the four most common mutations account for 78% of mutant alleles in the glucose-6-phosphatase (G6Pase) gene. A significant fraction of mutant alleles remain unidentified. Thus, informative polymorphic markers are necessary for linkage analysis in carrier testing and prenatal diagnosis in families where mutations can not be identified. The common mutations appear to be ethnic-specific, suggesting that the individual mutations may have a common founder. With the recent discovery of the nucleotide 1176 polymorphism, we have studied whether these mutations are in linkage disequilibrium with the polymorphism. The results of polymerase chain reaction/allele-specific oligonucleotide analysis show that nucleotide 1176 C is in linkage disequilibrium with mutations R83 C and R83H, and with the splicing mutation 727G→T. The 1176 T polymorphism is in linkage disequilibrium with 459insTA. A GT repeat polymorphism has also been found. However, its heterozygosity is low. The 1176 nucleotide polymorphic marker can be used in carrier and prenatal diagnosis of GSD1a families that have unidentified mutations and are informative for this marker. Received: 27 January 1998 / Accepted: 17 April 1998     

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.