Human Mutation

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, the relevance of rare exonic single‐base substitutions at nucleotide positions close to splice sites that are predicted to result in missense or silent (SNP) variants or substitutions in introns at splice‐site positions that are not highly conserved has not been systematically examined in FAP patients. In 34 index patients, we identified 26 different heterozygous single‐base substitutions at or close to the splice sites. We characterized five exonic mutations in exon 4 (c.423G>T), exon 14 (c.1956C>T, c.1957A>G, and c.1957A>C), and exon 15 (c.1959G>A) by transcript analysis and by splice‐prediction programs (BDGP, SpliceSiteFinder, and ESEfinder). The splicing patterns of these variants were compared to those of 16 different substitutions at highly or less‐conserved intronic splice‐site positions, and to normal controls. In addition, we analyzed cosegregation of the variants with affected family members and examined the genotype–phenotype correlation. We could demonstrate that the four unclear variants in exon 4 and 14 that are predicted to result in missense or silent mutations in fact lead to complete exon skipping due to aberrant splicing; one possible explanation for this observed effect might be the disruption of exonic splicing enhancer (ESE) motifs. In contrast, the substitution at the first position of exon 15 seems to actually be a silent variant. We present the first systematic evaluation of different single‐base substitutions in APC at or close to splice sites at transcript level. We show that the consequence of exonic mutations cannot be evaluated only by the predicted change in amino acid sequence but rather by the change at DNA level. The functional analysis of variants with unknown pathogenic effect plays an important role in increasing the mutation detection rate and achieving validation of predictive testing. Hum Mutat 24:370–380, 2004. © 2004 Wiley‐Liss, Inc.

A subtype of phenylalanine hydroxylase (PAH) deficiency that responds to cofactor (tetrahydrobiopterin, BH4) supplementation has been associated with phenylketonuria (PKU) mutations. The underlying molecular mechanism of this responsiveness is as yet unknown and requires a detailed in vitro expression analysis of the associated mutations. With this aim, we optimized the analysis of the kinetic and cofactor binding properties in recombinant human PAH and in seven mild PKU mutations, i.e., c.194T>C (p.I65T), c.204A>T (p.R68S), c.731C>T (p.P244L), c.782G>A (p.R261Q), c.926C>T (p.A309V), c.1162G>A (p.V388M), and c.1162G>A (p.Y414C) expressed in E. coli. For p.I65T, p.R68S, and p.R261Q, we could in addition study the equilibrium binding of BH4 to the tetrameric forms by isothermal titration calorimetry (ITC). All the mutations resulted in catalytic defects, and p.I65T, p.R68S, p.P244L, and most probably p.A309V, showed reduced binding affinity for BH4. The possible stabilizing effect of the cofactor was explored using a cell‐free in vitro synthesis assay combined with pulse‐chase methodology. BH4 prevents the degradation of the proteins of folding variants p.A309V, p.V388M, and p.Y414C, acting as a chemical chaperone. In addition, for wild‐type PAH and all mild PKU mutants analyzed in this study, BH4 increases the PAH activity of the synthesized protein and protects from the rapid inactivation observed in vitro. Catalase and superoxide dismutase partially mimic this protection. All together, our results indicate that the response to BH4 substitution therapy by PKU mutations may have a multifactorial basis. Both effects of BH4 on PAH, i.e., the chemical chaperone effect preventing protein misfolding and the protection from inactivation, may be relevant mechanisms of the responsive phenotype. Hum Mutat 24:388–399, 2004. © 2004 Wiley‐Liss, Inc.

doi: 10.1002/humu.20096pmid: 15459955

Wilms tumor (WT) is a kidney malignancy of childhood characterized by highly heterogeneous genetic alterations. We previously reported the molecular and cytogenetic characterization of a WT (Case 30) carrying an interstitial deletion in chromosome 7p14 between markers D7S555 and D7S668. Loss of heterozygosity (LOH) analyses had revealed that this same region was lost in 8 out of 38 examined WTs, suggesting that the identified interval contains a putative tumor suppressor gene. To confirm this hypothesis, in this work, we analyzed an additional 35 WTs, four of which showed LOH in the region of interest. Furthermore, we were able to more accurately define the extension of the deletion in Case 30, mapping it within an interval not exceeding 390 kb, proximally to D7S555. To date, only a single expressed gene, POU6F2 (the POU domain, class 6, transcription factor 2; also known as RPF1), has been recognized in this interval. Sequencing of the gene in the 12 WTs showing LOH and in a corresponding numbers of WT cases without LOH, led to the identification of two germline nucleotide substitutions. The first occurred in the 5′‐untranslated region, while the second caused an amino acid change in a glutamine repeat domain. These mutations, whose occurrence was not observed in more than 100 control subjects, were detected in two patients showing the loss of the constitutionally wild‐type allele in tumor DNA. Together with the finding of the expression of the POU6F2 mouse homolog in both fetal and adult kidney, our observations suggest that the gene is a tumor suppressor and is involved in hereditary predisposition to WT. Hum Mutat 24:400–407, 2004. © 2004 Wiley‐Liss, Inc.

Human leukocyte antigen (HLA) class I genes present some of the most complex single nucleotide polymorphism (SNP) patterns in the human genome. HLA typing is therefore extremely challenging. In this article, we use the ligation detection reaction (LDR) combined with a universal array (UA) as a robust and efficient method to analyze SNPs within the HLA‐A region that includes HLA‐A alleles of interest for immunotherapy in tumor diseases. The LDR, combined with a UA platform, has been optimized for the detection of 27 alleles distributed within exons 2 and 3 of HLA‐A. The assay involves the amplification by PCR of the HLA‐A genomic region (1,900 bp), the cycled ligation reaction, followed by the capture of ligated products through hybridization onto a UA. Each slide was designed to allow the detection of up to eight samples in parallel. The PCR/LDR/UA HLA‐A assay was evaluated by analyzing 62 individuals (31 homozygous and 31 heterozygous) previously typed by direct sequencing. We demonstrate that the microarray genotyping procedure described here is a robust and efficient method for unambiguous detection of HLA alleles. HLA genotyping by PCR/LDR/UA is in perfect agreement with typing obtained by direct sequencing. Our results clearly demonstrate that the combination of enzymatic processing (LDR) and a demultiplexing hybridization onto a UA is a robust tool for SNP discrimination within the highly polymorphic HLA region. We demonstrate the specificity and efficiency of such an approach, suggesting the feasibility of a PCR/LDR/UA low resolution HLA typing procedure. Hum Mutat 24:428–434, 2004. © 2004 Wiley‐Liss, Inc.

doi: 10.1002/humu.20109pmid: N/A

The original article to which this Erratum refers was published in Human Mutation 24:215–224 Human Mutation(2004) 24(3) 215–224