Genes for familial hemiplegic migraine (FHM) and episodic ataxia type-2 (EA-2) have been mapped to chromosome 19p13. We characterized a brain-specific P/Q-type Ca2+ channel alpha1-subunit gene, CACNL1A4, covering 300 kb with 47 exons. Sequencing of all exons and their surroundings revealed polymorphic variations, including a (CA)n-repeat (D19S1150), a (CAG)n-repeat in the 3'-UTR, and different types of deleterious mutations in FHM and EA-2. In FHM, we found four different missense mutations in conserved functional domains. One mutation has occurred on two different haplotypes in unrelated FHM families. In EA-2, we found two mutations disrupting the reading frame. Thus, FHM and EA-2 can be considered as allelic channelopathies. A similar etiology may be involved in common types of migraine.

The Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome with facial abnormalities, broad thumbs, broad big toes and mental retardation as the main clinical features. Many patients with RTS have been shown to have breakpoints in, and microdeletions of, chromosome 16p13.3 (refs 4-8). Here we report that all these breakpoints are restricted to a region that contains the gene for the human CREB binding protein (CBP), a nuclear protein participating as a co-activator in cyclic-AMP-regulated gene expression. We show that RTS results not only from gross chromosomal rearrangements of chromosome 16p, but also from point mutations in the CBP gene itself. Because the patients are heterozygous for the mutations, we propose that the loss of one functional copy of the CBP gene underlies the developmental abnormalities in RTS and possibly the propensity for malignancy.

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Only a small proportion of cancers result from familial cancer syndromes with Mendelian inheritance. Nonfamilial, 'sporadic' cancers, which represent most cancer cases, also have a significant hereditary component, but the genes involved have low penetrance and are extremely difficult to detect. Therefore, mapping and cloning of quantitative trait loci (QTLs) for cancer susceptibility in animals could help identify homologous genes in humans. Several cancer-susceptibility QTLs have been mapped in mice and rats, but none have been cloned so far. Here we report the positional cloning of the mouse gene Scc1 (Susceptibility to colon cancer 1) and the identification of Ptprj, encoding a receptor-type protein tyrosine phosphatase, as the underlying gene. In human colon, lung and breast cancers, we show frequent deletion of PTPRJ, allelic imbalance in loss of heterozygosity (LOH) and missense mutations. Our data suggest that PTPRJ is relevant to the development of several different human cancers.

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

Despite the presence of intracellular pathogen recognition receptors that allow infected cells to attract the immune system, undifferentiated keratinocytes (KCs) are the main targets for latent infection with high-risk human papilloma viruses (hrHPVs). HPV infections are transient but on average last for more than one year suggesting that HPV has developed means to evade host immunity. To understand how HPV persists, we studied the innate immune response of undifferentiated human KCs harboring episomal copies of HPV16 and 18 by genome-wide expression profiling. Our data showed that the expression of the different virus-sensing receptors was not affected by the presence of HPV. Poly(I:C) stimulation of the viral RNA receptors TLR3, PKR, MDA5 and RIG-I, the latter of which indirectly senses viral DNA through non-self RNA polymerase III transcripts, showed dampening in downstream signalling of these receptors by HPVs. Many of the genes downregulated in HPV-positive KCs involved components of the antigen presenting pathway, the inflammasome, the production of antivirals, pro-inflammatory and chemotactic cytokines, and components downstream of activated pathogen receptors. Notably, gene and/or protein interaction analysis revealed the downregulation of a network of genes that was strongly interconnected by IL-1β, a crucial cytokine to activate adaptive immunity. In summary, our comprehensive expression profiling approach revealed that HPV16 and 18 coordinate a broad deregulation of the keratinocyte's inflammatory response, and contributes to the understanding of virus persistence.

Currently available techniques used to recognize point mutations in genetic disease are time consuming and are capable of screening only small pieces of DNA. Moreover, they detect all sequence differences including phenotypically silent changes. Consequently, they are not convenient to analyse mutations in large, multi-exonic genes, where a large fraction of pathological point mutations arises from early termination, as is the case for the one third non-deletion/duplication cases of Duchenne Muscular Dystrophy. We have developed a rapid and sensitive method, the Protein Truncation Test (PTT). PTT is based on a combination of RT-PCR, transcription and translation and selectively detects translation-terminating mutations. We demonstrate its effectiveness to detect point mutations in DMD-patients and carrier females. PTT should be widely applicable diagnostically in any disease where early terminations contribute substantially to the disease cause.

To identify serum biomarkers that allow monitoring of disease progression and treatment effects in Duchenne muscular dystrophy (DMD) patients, levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1) and osteopontin (OPN) were determined in 63 DMD patients on corticosteroid therapy. These proteins were selected for their role in the pathogenesis of muscular dystrophy. Levels of MMP-9 and TIMP-1 were significantly higher in sera of DMD patients compared to healthy controls, whereas the OPN levels showed no significant difference. MMP-9 levels were also observed to be significantly higher in older, nonambulant patients, compared to ambulant patients. Longitudinal data from a smaller cohort of DMD patients followed up for over 4years showed that MMP-9, but not TIMP-1 increased significantly with age. Hence, MMP-9 is a potential DMD biomarker for disease progression. Future studies have to confirm whether serum MMP-9 levels can be used to monitor therapeutic response.