The analysis of a lymphoblastoid cell line (5106), derived from a rare individual of normal intelligence with an unmethylated full mutation of the FMR1 gene, allowed us to reconstruct the chain of molecular events leading to the FMR1 inactivation and to fragile X syndrome. We found that lack of DNA methylation of the entire promoter region, including the expanded CGG repeat, correlates with methylation of lysine 4 residue on the N-tail of histone H3 (H3-K4), as in normal controls. Normal levels of FMR1 mRNA were detected by real-time fluorescent RT-PCR (0.8-1.4 times compared with a control sample), but mRNA translation was less efficient (-40%), as judged by polysome profiling, resulting in reduced levels of FMRP protein (approximately 30% of a normal control). These results underline once more that CGG repeat amplification per se does not prevent FMR1 transcription and FMRP production in the absence of DNA methylation. Surprisingly, we found by chromatin immunoprecipitation that cell line 5106 has deacetylated histones H3 and H4 as well as methylated lysine 9 on histone H3 (H3-K9), like fragile X cell lines, in both the promoter and exon 1. This indicates that these two epigenetic marks (i.e. histone deacetylation and H3-K9 methylation) can be established in the absence of DNA methylation and do not interfere with active gene transcription, contrary to expectation. Our results also suggest that the molecular pathways regulating DNA and H3-K4 methylation are independent from those regulating histone acetylation and H3-K9 methylation.

X-linked retinitis pigmentosa (XLRP) is genetically heterogeneous with two causative genes identified, RPGR and RP2. We previously mapped a locus for a severe form of XLRP, RP23, to a 10.71 Mb interval on Xp22.31-22.13 containing 62 genes. Candidate gene screening failed to identify a causative mutation, so we adopted targeted genomic next-generation sequencing of the disease interval to determine the molecular cause of RP23. No coding variants or variants within or near splice sites were identified. In contrast, a variant deep within intron 9 of OFD1 increased the splice site prediction score 4 bp upstream of the variant. Mutations in OFD1 cause the syndromic ciliopathies orofaciodigital syndrome-1, which is male lethal, Simpson-Golabi-Behmel syndrome type 2 and Joubert syndrome. We tested the effect of the IVS9+706A>G variant on OFD1 splicing in vivo. In RP23 patient-derived RNA, we detected an OFD1 transcript with the insertion of a cryptic exon spliced between exons 9 and 10 causing a frameshift, p.N313fs.X330. Correctly spliced OFD1 was also detected in patient-derived RNA, although at reduced levels (39%), hence the mutation is not male lethal. Our data suggest that photoreceptors are uniquely susceptible to reduced expression of OFD1 and that an alternative disease mechanism can cause XLRP. This disease mechanism of reduced expression for a syndromic ciliopathy gene causing isolated retinal degeneration is reminiscent of CEP290 intronic mutations that cause Leber congenital amaurosis, and we speculate that reduced dosage of correctly spliced ciliopathy genes may be a common disease mechanism in retinal degenerations.

Community (QLG2-CT-1999-00988) (to FM), CAICYT of Spanish Ministerio de Ciencia y Tecnología (SAF99-0025) (to FM), and Fondo de Investigaciones Sanitarias (FIS 00/0244) (to IdC).

Hereditary nonpolyposis colorectal cancer (HNPCC) is a cancer-predisposing condition caused by inactivating mutations in at least four genes (MSH2, MLH1, MSH6, and PMS2) belonging to the mismatch repair system. At present, availability of the microsatellite instability (MSI) test allows screening of a relevant fraction of patients with a constellation of features suggestive of HNPCC. By analogy with several other genetic disorders, it is clearly emerging that the term HNPCC encompasses a wide spectrum of different clinical presentations, including Muir-Torre syndrome, Turcot syndrome, and the neurofibromatosis-hematological malignancy association. Notwithstanding the remarkable genetic and allelic heterogeneity, a few consistent phenotype-genotype associations can be recognized. Mutations in the MSH2 gene entail higher risks of developing cancer, including extraintestinal ones, than MLH1 alterations. MSH2 also accounts for most cases of Muir-Torre syndrome, which is characterized by the presence of sebaceous skin tumors. The few known PMS2 mutations show a striking association with the presence of gliomas, which are the hallmark of the Turcot variant of HNPCC. Homozygotes for mismatch repair gene mutations present with stigmata of neurofibromatosis 1 and usually die in childhood due to a variety of leukemias and lymphomas. While such correlations are being defined, the underlying reasons have only partially been elucidated, and may include heterogeneous gene functions and properties; types of mutation, some of which may exert dominant negative effects; and genetic and environmental modifiers.