Homozygous disruption of PDZD7 by reciprocal translocation in a consanguineous family: a new member of the Usher syndrome protein interactome causing congenital hearing impairment.

A homozygous reciprocal translocation, 46,XY,t(10;11),t(10;11), was detected in a boy with non-syndromic congenital sensorineural hearing impairment. Both parents and their four other children were heterozygous translocation carriers, 46,XX,t(10;11) and 46,XY,t(10;11), respectively. Fluorescence in situ hybridization of region-specific clones to patient chromosomes was used to localize the breakpoints within bacterial artificial chromosome (BAC) RP11-108L7 on chromosome 10q24.3 and within BAC CTD-2527F12 on chromosome 11q23.3. Junction fragments were cloned by vector ligation and sequenced. The chromosome 10 breakpoint was identified within the PDZ domain containing 7 (PDZD7) gene, disrupting the open reading frame of transcript PDZD7-C (without PDZ domain) and the 5'-untranslated region of transcript PDZD7-D (with one PDZ and two prolin-rich domains). The chromosome 11 breakpoint was localized in an intergenic segment. Reverse transcriptase-polymerase chain reaction analysis revealed PDZD7 expression in the human inner ear. A murine Pdzd7 transcript that is most similar in structure to human PDZD7-D is known to be expressed in the adult inner ear and retina. PDZD7 shares sequence homology with the PDZ domain-containing genes, USH1C (harmonin) and DFNB31 (whirlin). Allelic mutations in harmonin and whirlin can cause both Usher syndrome (USH1C and USH2D, respectively) and congenital hearing impairment (DFNB18 and DFNB31, respectively). Protein-protein interaction assays revealed the integration of PDZD7 in the protein network related to the human Usher syndrome. Collectively, our data provide strong evidence that PDZD7 is a new autosomal-recessive deafness-causing gene and also a prime candidate gene for Usher syndrome.

Identification and prevention of genotyping errors caused by G-quadruplex- and i-motif-like sequences.

BACKGROUND: Reliable PCR amplification of DNA fragments is the prerequisite for most genetic assays. We investigated the impact of G-quadruplex- or i-motif-like sequences on the reliability of PCR-based genetic analyses. METHODS: We found the sequence context of a common intronic polymorphism in the MEN1 gene (multiple endocrine neoplasia I) to be the cause of systematic genotyping errors by inducing preferential amplification of one allelic variant [allele dropout (ADO)]. Bioinformatic analyses and pyrosequencing-based allele quantification enabled the identification of the underlying DNA structures. RESULTS: We showed that G-quadruplex- or i-motif-like sequences can reproducibly cause ADO. In these cases, amplification efficiency strongly depends on the PCR enzyme and buffer conditions, the magnesium concentration in particular. In a randomly chosen subset of candidate single-nucleotide polymorphisms (SNPs) defined by properties deduced from 2 originally identified ADO cases, we confirmed preferential PCR amplification in up to 50% of the SNPs. We subsequently identified G-quadruplex and i-motifs harboring a SNP that alters the typical motif as the cause of this phenomenon, and a genomewide search based on the respective motifs predicted 0.5% of all SNPs listed by dbSNP and Online Mendelian Inheritance in Man to be potentially affected. CONCLUSIONS: Undetected, the described phenomenon produces systematic errors in genetic analyses that may lead to misdiagnoses in clinical settings. PCR products should be checked for G-quadruplex and i-motifs to avoid the formation of ADO-causing secondary structures. Truly affected assays can then be identified by a simple experimental procedure, which simultaneously provides the solution to the problem.

Monozygotic twins discordant for constitutive BRCA1 promoter methylation, childhood cancer and secondary cancer.

We describe monozygotic twins discordant for childhood leukemia and secondary thyroid carcinoma. We used bisulfite pyrosequencing to compare the constitutive promoter methylation of BRCA1 and several other tumor suppressor genes in primary fibroblasts. The affected twin displayed an increased BRCA1 methylation (12%), compared with her sister (3%). Subsequent bisulfite plasmid sequencing demonstrated that 13% (6 of 47) BRCA1 alleles were fully methylated in the affected twin, whereas her sister displayed only single CpG errors without functional implications. This between-twin methylation difference was also found in irradiated fibroblasts and untreated saliva cells. The BRCA1 epimutation may have originated by an early somatic event in the affected twin: approximately 25% of her body cells derived from different embryonic cell lineages carry one epigenetically inactivated BRCA1 allele. This epimutation was associated with reduced basal protein levels and a higher induction of BRCA1 after DNA damage. In addition, we performed a genome-wide microarray analysis of both sisters and found several copy number variations, i.e., heterozygous deletion and reduced expression of the RSPO3 gene in the affected twin. This monozygotic twin pair represents an impressive example of epigenetic somatic mosaicism, suggesting a role for constitutive epimutations, maybe along with de novo genetic alterations in recurrent tumor development.