Quality of diagnostic mutation analyses for phenylketonuria.

DNA sequence analyses have become a major component in the diagnostic work-up of patients; however, limited consideration appears to be given to the possibility that reported results may in fact be wrong. Over the last four years we have carried out an External Quality Assessment scheme for mutation analysis in phenylketonuria. Each year, three DNA samples with previously characterized genotypes were mailed to participating laboratories. Indications for testing were either confirmation of diagnosis and prediction of disease severity, or carrier analysis. Each year there were several laboratories that failed to identify mutations because of methodological limitations. Of the participating laboratories that used comprehensive mutation detection methods, each year there was at least one that missed at least one mutation. Indeed, in the 2007 scheme almost 8% of reports from laboratories that used comprehensive mutation detection methods such as sequencing of all exons of the PAH gene contained incorrect genotypes. There were also serious deficiencies in the interpretation of genotype data: in the 2007 scheme, 6 out of 10 laboratories that obtained full genotyping marks for interpretation incurred a reduction of marks because information on the expected phenotype was missing or wrong. Several laboratories failed to appreciate the clinical relevance of a mutation associated with mild hyperphenylalaninaemia, which does not require treatment, and some discussed the option of prenatal diagnosis in the respective case. In conclusion, mutation analyses may be prone to errors and this demands careful interpretation of results in relation to clinical and biochemical findings.

Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens.

Congenital absence of the vas deferens (CAVD) is a frequent cause for obstructive azoospermia and accounts for 1%-2% of male infertility. A high incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has recently been reported in males with CAVD. We have investigated a cohort of 106 German patients with congenital bilateral or unilateral absence of the vas deferens for mutations in the coding region, flanking intron regions and promotor sequences of the CFTR gene. Of the CAVD patients, 75% carried CFTR mutations or disease-associated CFTR variants, such as the "5T" allele, on both chromosomes. The distribution of mutation genotypes clearly differed from that observed in cystic fibrosis. None of the CAVD patients was homozygous for delta F508 and none was compound heterozygous for delta F508 and a nonsense or frameshift mutation. Instead, homozygosity was found for a few mild missense or splicing mutations, and the majority of CAVD mutations were missense substitutions. Twenty-one German CAVD patients were compound heterozygous for delta F508 and R117H, which was the most frequent CAVD genotype in our study group. Haplotype analysis indicated a common origin for R117H in our population, whereas another frequent CAVD mutation, viz. the "5T allele" was a recurrent mutation on different intragenic haplotypes and multiple ethnic backgrounds. We identified a total of 46 different mutations and variants, of which 15 mutations have not previously been reported. Thirteen novel missense mutations and one unique amino-acid insertion may be confined to the CAVD phenotype. A few splice or missense variants, such as F508C or 1716 G-->A, are proposed here as possible candidate CAVD mutations with an apparently reduced penetrance. Clinical examination of patients with CFTR mutations on both chromosomes revealed elevated sweat chloride concentrations and discrete symptoms of respiratory disease in a subset of patients. Thus, our collaborative study shows that CAVD without renal malformation is a primary genital form of cystic fibrosis in the vast majority of German patients and links the particular expression of clinical symptoms in CAVD with a distinct subset of CFTR mutation genotypes.

Molecular analysis and genetic mapping of the rhodopsin gene in families with autosomal dominant retinitis pigmentosa.

Eighty-eight patients/families with autosomal dominant retinitis pigmentosa (RP) were screened for rhodopsin mutations. Direct sequencing revealed 13 different mutations in a total of 14 (i.e., 16%) unrelated patients. Five of these mutations (T4K, Q28H, R135G, F220C, and C222R) have not been reported so far. In addition, multipoint linkage analysis was performed on two large families with autosomal dominant RP due to rhodopsin mutations by using five DNA probes from 3q21-q24. No tight linkage was found between the rhodopsin locus (RHO) and D3S47 (theta max = 0.08). By six-point analysis, RHO was localized in the region between D3S21 and D3S47, with a maximum lod score of 13.447 directly at D3S20.

Heterogeneity of mutations in Bulgarian phenylketonuria haplotype 1 and 4 alleles.

A study of Bulgarian patients with classical PKU demonstrated that haplotypes 1 and 4 carry a significant number of rare molecular defects resulting from independent mutational events. Differences in mutations associated with these common haplotypes exist even between populations which share a common major PKU mutation. Some amino acid substitutions, previously reported to lead to mild phenylalanine hydroxylase deficiency, were detected in the present study in compound heterozygotes with severe PKU. These findings preclude carrier testing and hyperphenylalaninemia typing by genomic analysis at least in the heterogeneous Bulgarian population.

Analysis of exon 7 of the human phenylalanine hydroxylase gene: a mutation hot spot?

Complete sequence analysis of 194 human phenylalanine hydroxylase genes from PKU patients originating from West Germany and Bulgaria revealed 13 different mutations within exon 7 of the gene. Four of these mutations (T238P: ACT-->CCT; L242F:CTC-->TTC; R252G:CGG-->GGG; and 1043 delta 11: nt 1043-nt 1053 deleted) have so far not been described in the literature. Including these new mutations at least 21 different gene lesions and one sequence polymorphism exist for exon 7. Despite this large number unbiased calculation of the mutation frequency/exon size ratio does not provide conclusive evidence that exon 7 is a hot spot for disease causing mutations. Extensive screening during our experiments also failed to demonstrate the existence of excessive polymorphism in this part of the gene. It might therefore be speculated that the functional importance of the highly conserved exon 7 sequence accounts for the clustering of observed mutations which result in clinically manifest PKU. In addition we report our experience in regard to the resolution capacity of denaturing gradient gel electrophoresis (DGGE), a nonradioactive technique for the rapid screening of unknown mutations in exon 7.

Recurrent nonsense mutation in exon 7 of the phenylalanine hydroxylase gene.

A new mutation (CGA to TGA) in codon 261 of exon 7 of the phenylalanine hydroxylase gene transforms Arg261 to a stop codon in two unrelated patients of German and Turkish origin. The different ethnic backgrounds and the different polymorphic characteristics of the two mutant alleles suggest an independent origin of the mutation. This is the second defect detected in codon 261 of the phenylalanine hydroxylase gene, a codon that thus appears to be a mutation hot spot.

Silent mutations in the phenylalanine hydroxylase gene as an aid to the diagnosis of phenylketonuria.

Direct sequencing of the phenylalanine hydroxylase (PAH) gene indicated the existence of silent mutations in codons 232, 245, and 385, linked to specific RFLP haplotypes in several Caucasian populations, namely Germans, Bulgarians, Italians, Turks, and Lithuanians. All three mutations create a new restriction site and can be easily detected on PCR amplified DNA. The usefulness of the silent mutations for diagnostic purposes depends on the haplotype distribution in the target population. The combined analysis of these markers and one or two PKU mutations forms a simple panel of diagnostic tests with full informativeness in a large proportion of PKU families, which helps to avoid the problems of genetic heterogeneity and of prenatal genomic Southern blot analysis.

Aberrant splicing of phenylalanine hydroxylase mRNA: the major cause for phenylketonuria in parts of southern Europe.

We report a mutation within the phenylalanine hydroxylase (PAH) gene that causes aberrant splicing of the mRNA and that is in tight association with chromosomal haplotypes 6, 10, and 36. Because of the high frequency of these particular haplotypes in Bulgaria, Italy, and Turkey, it appears to be one of the more frequent defects in the PAH gene causing classical phenylketonuria in this part of Europe. The mutation is a G to A transition at position 546 in intron 10 of the PAH gene, 11 bp upstream from the intron 10/exon 11 boundary. It activates a cryptic splice site and results in an in-frame insertion of 9 nucleotides between exon 10 and exon 11 of the processed mRNA. Normal amounts of liver PAH protein are present in homozygous patients, but no catalytic activity can be detected. This loss of enzyme activity is probably caused by conformational changes resulting from the insertion of three additional amino acids (Gly-Leu-Gln) between the normal sequences encoded by exon 10 and exon 11.

Molecular analysis of PKU haplotypes in the population of southern Poland.

Out of a population of 138,598 infants born in southern Poland between 1987 and 1989, and screened for phenylketonuria (PKU), 28 cases were ascertained probands and their parents were isolated and eight polymorphic restriction sites were analyzed within the phenylalanine hydroxylase gene region. Twenty-one different haplotypes (HT) were revealed, five of them representing new categories. The most common haplotypes among those carrying normal alleles were: HT1 (27.3%) and HT4 (11.4%). Within the group of haplotypes with mutant alleles the most frequent was HT2 (56.8%), whereas the frequency of this haplotype in other European populations, such as French, Danish and German, ranged from 12% to 24%. HT3, being the most common in Danish (38%), and relatively frequent in the other western European (13-14%) populations, appeared to be very rare in our sample (2.3%). The mutation of codon 408 (exon 12, C----T, Arg----Trp), which has been described to be tightly linked to HT2, was tested on amplified DNA by dot-blot hybridization. This mutation was found in 25 out of 44 proband chromosomes. In one case it was linked to HT5, in the remaining 24 to HT2. Our results confirm molecular heterogeneity of PKU haplotypes, as well as their significant interpopulation variation.

Phenylalanine hydroxylase gene: novel missense mutation in exon 7 causing severe phenylketonuria.

By direct sequence analysis of 94 mutant phenylalanine hydroxylase alleles using polymerase chain reaction-based techniques, we identified a C to T transition in exon 7 of the human phenylalanine hydroxylase gene that is associated with RFLP haplotypes 1 and 4. A leucine for proline substitution at position 281 can be predicted from the nucleotide sequence of the mutant codon. Expression analysis in cultured mammalian cells after site-directed mutagenesis proved that the base substitution is a disease causing gene lesion. Dot-blot hybridization analysis using allele-specific oligonucleotides revealed that 25% of all mutant haplotype 1 alleles in the German population bear this mutation. In addition, this mutation could be detected on one mutant haplotype 4 allele. The fact that this mutation is associated with only 25% of all mutant haplotype 1 alleles suggests that multiple mutations may be associated with this haplotype. The occurrence of several different mutations would be in agreement with the clinical heterogeneity observed in the group of patients whose PKU alleles belong to haplotype 1.

Classical phenylketonuria in Bulgaria: RFLP haplotypes and frequency of the major mutations.

RFLP haplotypes and common mutations in the phenylalanine hydroxylase gene have been studied in a group of 29 Bulgarian PKU families. Haplotype distribution differs from that in other European populations, with a predominance of haplotypes 2 and 6 and a total absence of haplotype 3. The amino acid substitution in codon 408 is the most frequent molecular defect. The splicing defect in intron 12 is not found in Bulgarian PKU patients. Testing for three mutations, reported to be common among haplotype 1 and 4 alleles, has shown that they occur less frequently in Bulgarian PKU patients. Screening with five pairs of allele specific oligonucleotides failed to show the mutation in 59% of the patients. These findings add to the evidence that PKU is heterogeneous and that significant interpopulation differences exist. At present, DNA data cannot be used as an aid in early clinical classification and prognosis of hyperphenylalaninaemia in Bulgaria.

Phenylalanine hydroxylase gene: silent mutation uncovers evolutionary origin of different alleles.

Analyzing a panel of 94 phenylketonuria (PKU) alleles for mutations within the phenylalanine hydroxylase (PAH) gene, we identified a G to A transition in exon 7 corresponding to nucleotide 957 in the cDNA sequence. This nucleotide substitution generates a new Alu I site (...GTGGCT...----...GTAGCT...), but does not change the encoded amino acid (GTG245----GTA245 = VAL). In our panel of patients the Alu I polymorphism is exclusively associated with haplotypes 4 (mutant or normal alleles) and 3, 16, 17, 28 (normal alleles).

Hb D Los Angeles (D-Punjab) and Hb Presbyterian: analysis of the defect at the DNA level.

Amplification of the beta-globin gene by the polymerase chain reaction (PCR) and direct sequencing were used for a fast and reliable identification of the beta-globin variant Hb D Los Angeles and revealed the predicted G----C substitution in codon 121. The same method showed the molecular defect in Hb Presbyterian to be a C----G substitution in codon 108; this eliminates a MaeII restriction site.

Molecular basis of beta-thalassemia in Turkey: detection of rare mutations by direct sequencing.

Using restriction endonuclease analysis, oligonucleotide hybridization, and direct sequencing of amplified genomic DNA, we characterized 11 different mutations in the DNA of 26 patients from Turkey homozygous for beta-thalassemia. We found that mutations IVS-1 nt110, IVS-1 nt6, and the frameshift at codon 8 were the most frequent. By direct sequencing we characterized two very rare mutations not previously reported in the Turkish population: a frameshift +1 at codons 9/10 and a nonsense mutation at codon 15.

Phenylketonuria: detection of a frequent haplotype 4 allele mutation.

By sequence analysis of 94 phenylketonuria (PKU) alleles using polymerase chain reaction (PCR) based techniques, we identified a G to A transition in exon 5 of the human phenylalanine hydroxylase gene. This base substitution predicts an Arg158----Glu158 amino acid exchange and is strongly associated with the mutant haplotype 4 PKU allele.

A beta-thalassemia gene caused by a 290-base pair deletion: analysis by direct sequencing of enzymatically amplified DNA.

The base composition around a recently detected deletion in the human beta-globin gene was determined by direct DNA sequencing of an enzymatically amplified DNA segment. The deletion removes 290 base pairs (bp), including the entire exon 1 and the mRNA cap site. In the vicinity of the deletion endpoints, the normal beta-globin gene contains direct and inverted repeats which may have taken part in generation of this deletion.