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Huntington's disease is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat whose length is the major determinant of age at onset but remaining variation appears to be due in part to the effect of genetic modifiers. GRIK2, which encodes GluR6, a mediator of excitatory neurotransmission in the brain, has been suggested in several studies to be a modifier gene based upon a 3′ untranslated region TAA trinucleotide repeat polymorphism. Prior to investing in detailed studies of the functional impact of this polymorphism, we sought to confirm its effect on age at onset in a much larger dataset than in previous investigations. We genotyped the HD CAG repeat and the GRIK2 TAA repeat in DNA samples from 2,911 Huntington's disease subjects with known age at onset, and tested for a potential modifier effect of GRIK2 using a variety of statistical approaches. Unlike previous reports, we detected no evidence of an influence of the GRIK2 TAA repeat polymorphism on age at motor onset. Similarly, the GRIK2 polymorphism did not show significant modifier effect on psychiatric and cognitive age at onset in HD. Comprehensive analytical methods applied to a much larger sample than in previous studies do not support a role for GRIK2 as a genetic modifier of age at onset of clinical symptoms in Huntington's disease.

Huntington’s disease (HD) is an inherited neurodegenerative disorder characterized by motor, cognitive and behavioral disturbances, caused by the expansion of a CAG trinucleotide repeat in the HD gene. The CAG allele size is the major determinant of age at onset (AO) of motor symptoms, although the remaining variance in AO is highly heritable. The rs7665116 SNP in PPARGC1A, encoding the mitochondrial regulator PGC-1α, has been reported to be a significant modifier of AO in three European HD cohorts, perhaps due to affected cases from Italy. We attempted to replicate these findings in a large collection of (1,727) HD patient DNA samples of European origin. In the entire cohort, rs7665116 showed a significant effect in the dominant model (p value = 0.008) and the additive model (p value = 0.009). However, when examined by origin, cases of Southern European origin had an increased rs7665116 minor allele frequency (MAF), consistent with this being an ancestry-tagging SNP. The Southern European cases, despite similar mean CAG allele size, had a significantly older mean AO (p

Background & Aims To prospectively assess the diagnostic accuracy of the incorporation of additional magnetic resonance imaging (MRI) parameters in those based on contrast enhancement pattern for the diagnosis of solitary nodules between 5 and 20mm, detected during surveillance in patients with cirrhosis. Methods Between November 2003 and January 2010, we prospectively included 159 cirrhotic patients with a newly detected solitary nodule between 5 and 20mm in diameter by screening ultrasonography (US). Hepatic MRI and fine-needle biopsy were performed in all patients. Results Final diagnoses were hepatocellular carcinoma (HCC) (n=103), other malignant lesions (intrahepatic cholangiocarcinoma/metastases) (n=4), and benign lesions (n=52). The specific enhancement pattern (arterial enhancement followed by washout) yielded a sensitivity and specificity of 58.3% and 96.4%, respectively. Peritumoral capsule was present in 43 HCC and in 2 non-HCC lesions. Intralesional fat was detected in 24 nodules; 5 nodules were non-HCC. Finally, the presence of both capsule and fat was observed in 10 cases, all of them HCC (100% specificity), but all of them also displayed the specific enhancement pattern, thus adding no sensitivity or specificity. Conclusions Conclusive non-invasive diagnosis of HCC in cirrhosis should be based only on the contrast enhancement pattern, while other characteristics at MRI do not increase the diagnostic accuracy.

Background & Aims Transarterial chemoembolisation (TACE) improves survival of properly selected patients with hepatocellular carcinoma (HCC). Drug eluting beads (DEB) provide a calibrated and homogenous procedure while increasing efficacy. Outcome data applying this technology is lacking, and this is instrumental for clinical decision-making and for trial design. We evaluated the survival of HCC patients treated with DEB-TACE following a strict selection (preserved liver function, absence of symptoms, extrahepatic spread or vascular invasion). Methods We registered baseline characteristics, the development of treatment-related adverse events, and the overall survival of all HCC patients treated by DEB-TACE from February 2004 to June 2010. Results One hundred and four patients were treated with DEB-TACE. All but one were cirrhotic, 62.5% HCV+, 95% Child-Pugh A, 41 BCLC-A and 63 BCLC-B. Causes of DEB-TACE treatment in BCLC-A patients were: 35 unfeasible ablation, and six post-treatment recurrences. After a median follow-up of 24.5months, 38 patients had died, two patients had received transplantation and 24 had received sorafenib because of untreatable tumour progression. Median survival of the cohort was 48.6months (95% CI: 36.9–61.2), while it was 54.2months in BCLC stage A and 47.7months in stage B. Median survival after censoring follow-up at time of transplant/sorafenib was 47.7 (95%CI: 37.9–57.5) months. Conclusions These data validate the safety of DEB-TACE and show that the survival expectancy applying current selection criteria and technique is better than that previously reported. A 50% survival at 4years should be considered when suggesting treatment for patients fitting into controversial scenarios such as expanded criteria for transplantation/resection for multifocal HCC.

Age at the onset of motor symptoms in Huntington disease (HD) is determined largely by the length of a CAG repeat expansion in HTT but is also influenced by other genetic factors. We tested whether common genetic variation near the mutation site is associated with differences in the distribution of expanded CAG alleles or age at the onset of motor symptoms. To define disease-associated single-nucleotide polymorphisms (SNPs), we compared 4p16.3 SNPs in HD subjects with population controls in a case:control strategy, which revealed that the strongest signals occurred at a great distance from the HD mutation as a result of “synthetic association” with SNP alleles that are of low frequency in population controls. Detailed analysis delineated a prominent ancestral haplotype that accounted for ∼50% of HD chromosomes and extended to at least 938 kb on about half of these. Together, the seven most abundant haplotypes accounted for ∼83% of HD chromosomes. Neither the extended shared haplotype nor the individual local HTT haplotypes were associated with altered CAG-repeat length distribution or residual age at the onset of motor symptoms, arguing against modification of these disease features by common cis-regulatory elements. Similarly, the 11 most frequent control haplotypes showed no trans-modifier effect on age at the onset of motor symptoms. Our results argue against common local regulatory variation as a factor influencing HD pathogenesis, suggesting that genetic modifiers be sought elsewhere in the genome. They also indicate that genome-wide association analysis with a small number of cases can be effective for regional localization of genetic defects, even when a founder effect accounts for only a fraction of the disorder.

Resumen El tumor de Krukenberg se define como aquel tumor metastasico en ovario uni o bilateral que contiene cantidades importantes de celulas en anillo de sello y cuyo origen es principalmente digestivo. La incidencia de tumores metastasicos en ovario es muy pequena (1-5%); asimismo la coincidencia de una masa ovarica de caracteristicas malignas metastasicas con una gestacion es infima. Presentamos el caso de una mujer de 19 anos que tras el diagnostico de un adenocarcinoma mucinoso de colon, a los 18 meses presenta un tumor de Krukenberg en la semana 29 de gestacion. En un primer momento se le realizo cesarea junto a cirugia resectiva del tumor. En un segundo tiempo se le practico cirugia citorreductora asociada a quimioterapia intraperitoneal intraoperatoria hipertermica, encontrandose actualmente en remision clinica y en seguimiento por el servicio de oncologia.

Background and aims Conventional pathological analysis fails to achieve sufficient sensitivity and specificity for the diagnosis of hepatocellular carcinoma (HCC) in small nodules. Immunohistochemical staining for glypican 3 (GPC3), heat shock protein 70 (HSP70) and glutamine synthetase (GS) has been suggested to allow a confident diagnosis but no prospective study has established the diagnostic accuracy of this approach. The aim of this study is to assess prospectively the diagnostic accuracy of a panel of markers (GPC3, HSP70, GS) for the diagnosis of HCC in patients with cirrhosis with a small (5–20 mm) nodule detected by ultrasound screening. Methods Sixty patients with cirrhosis with a single nodule 5–20 mm newly detected by ultrasound were included in the study. Contrast-enhanced ultrasound, magnetic resonance and fine needle biopsy of the nodule (gold standard) were performed; the biopsy was repeated in case of diagnostic failures. Three consecutive sections of the first biopsy sample with meaningful material were stained with antibodies against GPC3, HSP70 and GS. Results Forty patients were diagnosed with HCC. The sensitivity and specificity for HCC diagnosis were: GPC3 57.5% and 95%, HSP70 57.5% and 85%, GS 50% and 90%, respectively. The sensitivity and specificity of the different combinations were: GPC3+HSP70 40% and 100%; GPC3+GS 35% and 100%; HSP70+GS 35% and 100%; GPC3+HSP70+GS 25% and 100%. When at least two of the markers were positive (regardless of which), the sensitivity and specificity were 60% and 100%, respectively. Conventional pathological analysis yielded three false negative results, but the addition of this panel only correctly classified one of these cases as HCC. Conclusion These data within a prospective study establish the clinical usefulness of this panel of markers for the diagnosis of early HCC. However, the panel only slightly increases the diagnostic accuracy in an expert setting.

Objective To identify the genetic causes underlying early-onset autosomal recessive retinitis pigmentosa (arRP) in the Spanish population and describe the associated phenotype. Design Case series. Participants A total of 244 unrelated families affected by early-onset arRP. Methods Homozygosity mapping or exome sequencing analysis was performed in 3 families segregating arRP. A mutational screening was performed in 241 additional unrelated families for the p.Ser452Stop mutation. Haplotype analysis also was conducted. Individuals who were homozygotes, double heterozygotes, or carriers of mutations in RP1 underwent an ophthalmic evaluation to establish a genotype–phenotype correlation. Main Outcome Measures DNA sequence variants, homozygous regions, haplotypes, best-corrected visual acuity, visual field assessments, electroretinogram responses, and optical coherence tomography images. Results Four novel mutations in RP1 were identified. The new mutation p.Ser542Stop was present in 11 of 244 (4.5%) of the studied families. All chromosomes harboring this mutation shared the same haplotype. All patients presented a common phenotype with an early age of onset and a prompt macular degeneration, whereas the heterozygote carriers did not show any signs of retinitis pigmentosa (RP). Conclusions p.Ser542Stop is a single founder mutation and the most prevalent described mutation in the Spanish population. It causes early-onset RP with a rapid macular degeneration and is responsible for 4.5% of all cases. Our data suggest that the implication of RP1 in arRP may be underestimated. Financial Disclosure(s) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Summary Background Basal epidermolysis bullosa simplex (EBS) is a group of blistering geno-dermatoses mostly caused by mutations in the keratin genes, KRT5 and KRT14.Recessive mutations represent about 5% of all EBS mutations, being common andspecific in populations with high consanguinity, where affected patients showsevere phenotypes.Objectives To accomplish the first mutational analysis in patients of Spanish originwith EBS and to delineate a comprehensive genotype–phenotype correlation.Methods Twenty-one EBS families were analysed. Immunofluorescence mapping atthe dermoepidermal junction level was performed on skin biopsies from patients.Mutation screening of the entire coding sequences of KRT5 and KRT14 in genomicDNA was assessed by polymerase chain reaction and direct sequencing.Results KRT5 or KRT14 causative mutations were identified in 18 of the 21 EBSfamilies. A total of 14 different mutations were disclosed, of which 12 weredominant missense mutations and two truncating recessive mutations. Five of the14 mutations were novel including three dominant in KRT5 (p.V186E, p.T321Pand p.A428T) and two recessive in KRT14 (p.K116X and p.K250RfsX8). The twopatients with EBS carrying homozygous recessive mutations were affected bysevere phenotypes and belonged to consanguineous families. All five familieswith the EBS Dowling–Meara subtype carried recurrent mutations affecting thehighly conserved ends of the a-helical rod domain of K5 and K14. The sevenmutations associated with the localized EBS subtype were widely distributedalong the KRT5 and KRT14 genes. Two families with mottled pigmentation carriedthe P25L mutation in KRT5, commonly associated with this subtype.Conclusions This study further confirms the genotype–phenotype correlation estab-lished for EBS in other ethnic groups, and is the first in a Mediterranean country(excluding Israel). This study adds two novel recessive mutations to the worldwiderecord to date, which includes a total of 14 mutations. As in previous reports, therecessive mutations resulted in a lack of keratin K14, giving rise to a generalizedand severe presentation.

The use of magnetic resonance imaging (MRI) for assessment of Crohn's disease (CD) is expanding. The aim of this study is to define and provide an external validation of the MRI predictors of active CD, severe CD, and a quantitative Magnetic Resonance Index of Activity (MaRIA).In all, 48 patients with clinically active (n = 29) or inactive (n = 19) CD underwent ileocolonoscopy (reference standard) and MRI. T2-weighted and pre- and postcontrast-enhanced T1-weighted sequences were acquired. Endoscopic activity was evaluated by the Crohn's Disease Endoscopic Index of Severity (CDEIS), and also classified as absent, mild (inflammation without ulcers), or severe (presence of ulceration).In complete agreement with a previous derivation study, independent predictors of disease severity using CDEIS as a reference were wall thickness, relative contrast enhancement (RCE), presence of edema, and ulcers on MRI. Estimation of activity in each segment using this regression model, or another with simplified coefficients (MaRIA(S) = 1.5*wall thickness + 0.02*RCE + 5*edema + 10*ulceration) correlated with CDEIS (r = 0.798, P0.001; r = 0.80 P0.001, respectively). In the validation cohort both indexes had a high and equal accuracy for diagnosis of active disease: receiver operator characteristic (ROC) area 0.93, sensitivity 0.87, specificity 0.87 using a cutoff point ≥ 7, and for diagnosis of severe disease: ROC area 0.96, sensitivity 0.92, specificity 0.92 using a cutoff point ≥ 11. The total of segment values (MaRIA(T)) correlated with global CDEIS (r = 0.83, P0.001).The MRI variables that should be evaluated in clinical practice to diagnose active CD and severe CD are validated, as well as the quantitative index of activity for use in research studies.

Carmen Ayuso *, Juan Jose Telleria , Juan Carlos Tejedor c y Diego Gracia d a Servicio de Genetica, Fundacion Jimenez Diaz/CIBERER, Madrid, Espana b Laboratorio de Genetica, Instituto de Biologia y Genetica Molecular (IBGM), Universidad de Valladolid/CSIC, Valladolid, Espana c Servicio de Pediatria, Hospital Universitario de Mostoles, Mostoles, Madrid, Espana Departamento de Salud Publica e Historia de la Ciencia, Facultad de Medicina, Universidad Complutense, Madrid, Espana

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration that leads to progressive visual-field constriction and blindness. Although the disease manifests only in the retina, mutations in ubiquitously expressed genes associated with the tri-snRNP complex of the spliceosome have been identified in patients with dominantly inherited RP. We screened for mutations in PRPF6 (NM_012469.3), a gene on chromosome 20q13.33 encoding an essential protein for tri-snRNP assembly and stability, in 188 unrelated patients with autosomal-dominant RP and identified a missense mutation, c.2185C>T (p.Arg729Trp). This change affected a residue that is conserved from humans to yeast and cosegregated with the disease in the family in which it was identified. Lymphoblasts derived from patients with this mutation showed abnormal localization of endogenous PRPF6 within the nucleus. Specifically, this protein accumulated in the Cajal bodies, indicating a possible impairment in the tri-snRNP assembly or recycling. Expression of GFP-tagged PRPF6 in HeLa cells showed that this phenomenon depended exclusively on the mutated form of the protein. Furthermore, analysis of endogenous transcripts in cells from patients revealed intron retention for pre-mRNA bearing specific splicing signals, according to the same pattern displayed by lymphoblasts with mutations in other PRPF genes. Our results identify PRPF6 as the sixth gene involved in pre-mRNA splicing and dominant RP, corroborating the hypothesis that deficiencies in the spliceosome play an important role in the molecular pathology of this disease.

Purpose Heterozygous mutations around codon 838 of the guanylate cyclase 2D (GUCY2D) gene have recently been associated with more than a third of autosomal dominant macular dystrophy patients. The aim of our study was to evaluate the prevalence of these mutations in Spanish families with autosomal dominant cone, cone-rod, and macular dystrophies. Methods Mutation analysis was performed by PCR amplification of exon 13 of GUCY2D and subsequent restriction analysis. To confirm the results, automatic sequencing analysis was also performed. Results Among the 22 unrelated Spanish families included in the study, we found two associated disease mutations at codon 838 of the GUCY2D gene, one of which had not been previously described (p.R838P). This novel mutation exhibited phenotypic variability. Conclusions The prevalence of mutations around codon 838 of GUCY2D in our group of families (9.09%) is lower than that previously reported in other populations. However, the discovery of a novel mutation at codon 838 further suggests that this locus is a mutation hotspot within the GUCY2D gene, and confirms the importance of analyzing this codon to characterize molecularly these autosomal dominant retinal disorders.

Usher syndrome (USH) is an autosomal recessive disease characterized by hearing loss, retinitis pigmentosa (RP), and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous and is the most common cause underlying deafness and blindness of genetic origin. Clinically, USH is divided into three types. Usher type I (USH1) is the most severe form and is characterized by severe to profound congenital deafness, vestibular areflexia, and prepubertal onset of progressive RP. Type II (USH2) displays moderate to severe hearing loss, absence of vestibular dysfunction, and later onset of retinal degeneration. Type III (USH3) shows progressive postlingual hearing loss, variable onset of RP, and variable vestibular response. To date, five USH1 genes have been identified:MYO7A(USH1B),CDH23(USH1D),PCDH15(USH1F),USH1C(USH1C), andUSH1G(USH1G). Three genes are involved in USH2, namely,USH2A(USH2A),GPR98(USH2C), andDFNB31(USH2D). USH3 is rare except in certain populations, and the gene responsible for this type isUSH3A.

Resumen Fundamento y objetivo El sindrome de Holt-Oram (HOS) es una enfermedad de herencia autosomica dominante caracterizada por presentar defectos cardiovasculares y esqueleticos. El 85% aproximadamente de los afectos presentan mutaciones de novo en el gen TBX5. El objetivo de este estudio es proponer una estrategia molecular para diagnosticar a pacientes diagnosticados clinicamente de HOS. Pacientes y metodo Se secuencio del exon 2–8 del gen TBX5 a 7 pacientes. En aquellos sin mutacion identificada se empleo amplificacion de sondas dependientes de ligacion en multiplex (MLPA) utilizando las sondas p179 (ROR2, HOXD13, GLI3) y p180 (TBX5, SALL1, SALL4). Resultados En 2 casos, que cumplian los criterios estrictos descritos para HOS y presentaban antecedentes familiares de patologia similar, se identificaron las mutaciones p.Arg270X y p.Ala34Glyfsx27. En 3 casos se detecto mediante MLPA deleciones de la region codificante del gen GLI3. Conclusiones Para aumentar la tasa de deteccion mutacional en el gen TBX5 seria necesario realizar un examen fisico exhaustivo atendiendo a los criterios clinicos estrictos del sindrome, a fin de descartar otros sindromes con clinica solapantes a HOS. Asimismo, GLI3 podria ser un gen candidato a estudiar en los casos con sospecha clinica de HOS sin mutacion identificada en TBX5.

Hepatocellular Carcinoma: Diagnosis, staging, and treatment strategy Abstract Hepatocellular carcinoma is a tumor with a high incidence and high mortality. These data justify screening programs to enable curative treatments to improve survival rates. Screening the population at risk (mainly patients with cirrhosis of the liver) should include

Background: This study was undertaken to analyse the OA1 gene (GPR143) and its involvement in a Spanish family presenting with nystagmus, a common symptom of X-linked ocular albinism (XLOA). Methods: DNA samples from the index case and eight relatives were analysed by multiplex ligation-dependent probe amplification (MLPA). Sequence analysis and restriction assay were used to confirm the results. In addition, an analysis of a STR located in intron 1 of the OA1 gene (OA-CA) was performed. Results: The father of the proband presented with nystagmus, a feature consistent with XLOA. Mutation screening by multiplex ligation-dependent probe amplification and sequence analysis of the exon 2 of the OA1 gene led to the identification of the novel p.Glu129fsX35 (g.5815delA) mutation in two affected males and four carrier females. Three relatives were found to be non-mutated. The deletion detected resulted in a truncated protein 35 codons downstream and generated a new restriction site for the XcmI endonuclease. Additionally, microsatellite analysis showed co-segregation with the disease in the family. Conclusions: A novel deletion in the OA1 gene was identified in a Spanish family with ocular albinism. The mutation detected is likely a loss-of-function alteration. To the best of our knowledge, we describe the first Spanish family known to present with XLOA due to mutations in the OA1 gene.

Summary Background Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in COL7A1. The clinical manifestations are highly variable from nail dystrophy to life-threatening blistering, making early molecular diagnosis and prognosis of utmost importance for the affected families. Mutation identification is mandatory for prenatal testing. Objectives To conduct the first mutational analysis of COL7A1 in a Spanish cohort, to assess mutation consequences at protein/mRNA level and to establish genotype–phenotype correlations. Methods Forty-nine Spanish patients with DEB were studied. Antigen mapping was performed on patient skin biopsies. COL7A1 mutation screening in genomic DNA was performed by polymerase chain reaction (PCR) and direct sequencing. Mutation consequences were determined by reverse transcriptase-PCR. Results Eight patients belonged to three unrelated families with dominant DEB. Forty-one were affected with recessive DEB (RDEB). Specifically, 27 displayed the severe generalized subtype, eight the other generalized subtype and six a localized phenotype (two pretibial, three acral and one inversa). Thirty-five mutations were identified, 20 of which are novel. The pathogenic mutation c.6527insC accounted for 46·3% of Spanish RDEB alleles. A consistent genotype–phenotype correlation was established. Conclusions Although the COL7A1 database indicates that most DEB mutations are family specific, the pathogenic mutation c.6527insC was highly recurrent in our cohort. This level of recurrence for a single genetic defect has never previously been reported for COL7A1. Our findings are essential to the clinicians caring for patients with DEB in Spain and in the large population of Spanish descendants in Latin America. They also provide geneticists a molecular clue for a priority mutation screening strategy.

Hepatocellular carcinoma is a tumor with a high incidence and high mortality. These data justify screening programs to enable curative treatments to improve survival rates. Screening the population at risk (mainly patients with cirrhosis of the liver) should include ultrasonographic examination twice yearly. Given the vascular characteristics of hepatocellular carcinoma, it can be detected using dynamic techniques (contrast-enhanced ultrasonography, CT, and MRI). In cases in which the enhancement pattern is not characteristic, these techniques should be complemented with lesion biopsy. Once hepatocellular carcinoma is diagnosed, the tumor is staged, and together with the clinical condition of the patient, the stage will determine the most appropriate treatment strategy in each case.

Prenatal diagnosis with ultrasound findings compatible with skeletal dysplasia due to FGFR3 mutations over a 9 year period in pregnancies and abortuses. 54 samples were studied. Aneuploidy studies were carried out on all samples. By sequencing analysis, we determined mutations for achondroplasia (ACH), hypochondroplasia (HCH), and type I and type II tanathophoric dysplasia (TD). 2 chorionic villi samples had a G380R mutation due to a mother with ACH; 4 amniotic fluid samples with TDs in which the foetuses had micromelia plus hypoplastic thoraces; 5 samples from abortuses with TDs. Neither ACH nor HCH occurred in sporadic cases. Molecular studies in ongoing pregnancies are indicated in cases with an affected parent, a family history with positive molecular studies (maternal anxiety), and when the US finding demonstrates micromelia with a hypoplastic thorax. A protocol for tissues of abortuses should include an X-ray, pathologic anatomy, and genetic studies.

International audience; We report the results of molecular screening in 980 patients carried out as part of their work-up for suspected hereditary optic neuropathies. All the patients were investigated for Leber's hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA), by searching for the ten primary LHON-causing mtDNA mutations and examining the entire coding sequences of the OPA1 and OPA3 genes, the two genes currently identified in ADOA. Molecular defects were identified in 440 patients (45% of screened patients). Among these, 295 patients (67%) had an OPA1 mutation, 131 patients (30%) had an mtDNA mutation, and 14 patients (3%), belonging to three unrelated families, had an OPA3 mutation. Interestingly, OPA1 mutations were found in 157 (40%) of the 392 apparently sporadic cases of optic atrophy. The eOPA1 locus-specific database now contains a total of 204 OPA1 mutations, including 77 novel OPA1 mutations reported here. The statistical analysis of this large set of mutations has led us to propose a diagnostic strategy that should help with the molecular work-up of optic neuropathies. Our results highlight the importance of investigating LHON-causing mtDNA mutations as well as OPA1 and OPA3 mutations in cases of suspected hereditary optic neuropathy, even in absence of a family history of the disease.

Conventional colonoscopy combined with histologic analysis represents the standard of reference for the evaluation of colorectal disease and is usually the initial examination in patients with a suspected or established diagnosis of inflammatory bowel disease (IBD). However, it is increasingly being recognized that colonoscopy is limited to providing information regarding mucosal alterations. Colonoscopy cannot help estimate the depth of involvement of colonic lesions and does not provide information regarding the presence of extraluminal complications such as abscesses or fistulas. Recent technologic advances in magnetic resonance (MR) imaging, with its high spatial and tissue resolution, have raised expectations as to the potential role of this modality in the evaluation of colonic lesions in patients with IBD, as either a complement or an alternative to colonoscopy. MR colonography allows the characterization of colonic changes in acute and chronic IBD and can depict a wide spectrum of related lesions, including ulcers, edema, wall thickening, hyperemia, and fistulas, as well as potential extraluminal complications. The bulk of available evidence indicates that MR colonography can be useful as a problem-solving tool in the evaluation of IBD, as an alternative to colonoscopy whenever tissue sampling is not required, and for the assessment of the entire colon in cases of incomplete colonoscopy.

Background and purpose: The presence of cell-free fetal DNA in maternal plasma could allow performing a non-invasive prenatal diagnosis of Huntington disease (HD). The great advantage of this diagnosis is the absence of risk of fetal loss that it entails. Methods: Maternal plasma from four pregnant women in their first trimester of gestation with a fetus at-risk was studied. In all the four cases, the father was affected. Results: The diagnosis was performed both by a direct study of the mutation and an indirect haplotype study. By the direct analysis, three out of the four fetuses could be correctly diagnosed whilst the indirect analysis was only conclusive in one case. Conclusions: Non-invasive prenatal diagnosis of HD is possible by the analysis of fetal DNA in maternal plasma. Direct analysis of the mutation has shown higher accuracy than the haplotype analysis except for long expansions. Haplotype analysis would need to be improved for the study of Juvenile-onset HD. This diagnostic method would be limited to those couples with an affected male however this situation represents 80–90% of the pregnancies at-risk of HD. Moreover, it could be used as a confirmation test of healthy embryos transferred on pre-implantation genetic studies of HD.

Background/aims: Mutations in ABCA4 have been associated with autosomal recessive Stargardt disease (STGD), a few cases with autosomal recessive cone–rod dystrophy (arCRD) and autosomal recessive retinitis pigmentosa (arRP). The purpose of the study was threefold: to molecularly characterise families with no mutations or partially characterised families; to determine the specificity and sensitivity of the genotyping microarray; and to evaluate the efficiency of different methodologies. Methods: 23 STGD, five arCRD and three arRP Spanish patients who were previously analysed with the ABCR400 microarray were re-evaluated. Results were confirmed by direct sequencing. In patients with either none or only one mutant allele, ABCA4 was further analysed by denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA). Haplotype analysis was also performed. Results: In the first analysis performed with the microarray, 27 ABCA4 variants (27/62; 43.5%) were found. By dHPLC scanning, 12 novel mutations were additionally identified. In addition, two previously described mutations, one false negative (1/62; 1.6%) and one false positive (1.6%), were detected. MLPA analysis did not reveal additional substitutions. The new strategy yielded an increment of 21% compared with the approach used in the first round. Conclusion: ABCA4 should be analysed by optimal combination of high-throughput screening techniques such as microarray, dHPLC and direct sequencing. To the best of our knowledge, this strategy yielded significant mutational spectrum identification in Spanish patients with ABCA4 -associated phenotypes. Follow-up of patients, presenting an early onset of the disease and severe mutations, seems essential to perform accurate genotype–phenotype correlations and further characterisation of pathological ABCA4 alleles.

Severe ocular malformations, including anophthalmia-microphthalmia (AM), are responsible for around 25% of severe visual impairment in childhood. Recurrent interstitial deletions of 14q22–23 are associated with AM and a wide range of extra-ocular phenotypes including brain anomalies. The homeobox gene OTX2 is located at 14q22.3 and has recently been identified as mutated in AM patients. Eight human OTX2 mutations have been reported in subjects with severe eye malformations, including AM, and variable developmental delay. We screened a novel AM cohort for mutations and deletions in OTX2, and identified four new mutations in six individuals and two cases of whole gene deletions. Our data suggest that OTX2 mutations and deletions account for 2–3% of AM cases. © 2008 Wiley-Liss, Inc.

Background/aims: To determine the carrier frequency of ABCA4 mutations in order to achieve an insight into the prevalence of autosomal recessive Stargardt disease (arSTGD) in Spanish population. Methods: 133 arSTGD patients were analysed using the ABCR400 microarray and sequencing. Controls were analysed by two different strategies: 200 individuals were screened for the p.Arg1129Leu mutation by dHPLC and sequencing; whereas 78 individuals were tested for variants on the microarray and sequencing. Results: For the 1st strategy in controls, the p.Arg1129Leu variant was found in two heterozygous individuals, which would suppose a carrier frequency for any variant of ~6.0% and a calculated arSTGD prevalence of 1:1000. For the 2nd strategy, carrier frequency was 6.4% and therefore, an estimated prevalence of the disease of 1:870. Conclusion: Calculated prevalence of arSTGD, based on the ABCA4 carrier frequency, could be considerably higher than previous estimation. This discrepancy between observed (genotypic) and estimated (phenotypic) prevalence could be due to the existence of non-pathological or low penetrance alleles, which may result in late onset arSTGD or may be implicated in age-related macular degeneration (AMD). This situation should be regarded with especial care when genetic counselling is given and further follow-up of these patients should be recommended.

The role of contrastenhanced liver MRI using the hepatocyte-specific agent gadoliniumethoxybenzyl-diethylene triamine penta-acetic acid (Gd-EOB-DTPA, Primovist, Bayer Schering Pharma, Berlin, Germany) is considered, based on the discussion at the Forum of Liver MRI: The 1st International Primovist User Meeting. Current clinical evidence supports Gd-EOB-DTPA’s use in non-invasive diagnosis of benign and malignant liver lesions and staging of focal and diffuse liver disease. The information thus gained assists in the provision of cost-effective patient management. The pharmacological properties of Gd-EOB-DTPA facilitate the imaging of both the vascular and hepatobiliary phases in the cirrhotic and non-cirrhotic liver and may provide an opportunity for one-stop diagnosis of liver lesions, such as focal nodular hyperplasia, adenomas, haemangiomas, hepatocellular carcinoma and metastases. The currently recommended protocols using Gd-EOB-DTPA are appropriate for the majority of patients, but imaging time points may require modification in specific patient groups. Further experience in routine clinical practice, as opposed to that gained from rigidly designed clinical studies, may provide greater understanding of the potential of Gd-EOB-DTPA for the evaluation of the cirrhotic and non-cirrhotic liver. Additional studies may substantiate the potential of Gd-EOB-DTPA in the imaging of other abdominal organs and in the evaluation of liver function and fibrosis.

Ductal adenocarcinoma is the most common tumor of the pancreas, accounting for about 80% of all pancreatic tumors. The other 20% of pancreatic tumors is represented by a heterogeneous group of pancreatic neoplasms that includes cystic pancreatic neoplasms, islet cell tumors, and the so-called rare pancreatic tumors. In addition, the pancreatic gland may present a variety of inflammatory and pseudotumoral lesions that may mimic a primary pancreatic neoplasm. These uncommon tumors and pseudotumoral lesions present a wide spectrum of imaging findings and they are often poorly understood by the radiologist, becoming a diagnostic challenge. Some of these lesions may show an appearance similar to ductal adenocarcinoma being radiologically indistinguishable. However, some of these lesions sometimes may present specific features on imaging studies that may help to characterize the mass and to suggest a correct diagnosis. Many of these uncommon tumors and pseudotumoral lesions have a different approach, therapy, and prognosis than ductal adenocarcinoma. Therefore, it is important for the radiologist to be familiar with these entities to include them in the differential diagnosis to initiate an appropriate lesion-specific workup and treatment. In the present article, we review the radiological features of uncommon pancreatic tumors, atypical manifestations of ductal adenocarcinoma, and pseudotumoral masses, focusing on those features that can be helpful for the differential diagnosis.

Mutations within the inosine 5'-monophosphate dehydrogenase 1 (IMPDH1) gene cause the RP10 form of autosomal dominant retinitis pigmentosa (adRP), an early-onset retinopathy resulting in extensive visual handicap owing to progressive death of photoreceptors. Apart from the prevalence of RP10, estimated to account for 5-10% of cases of adRP in United States and Europe, two observations render this form of RP an attractive target for gene therapy. First, we show that while recombinant adeno-associated viral (AAV)-mediated expression of mutant human IMPDH1 protein in the mouse retina results in an aggressive retinopathy modelling the human counterpart, expression of a normal human IMPDH1 gene under similar conditions has no observable pathological effect on retinal function, indicating that over-expression of a therapeutic replacement gene may be relatively well tolerated. Secondly, complete absence of IMPDH1 protein in mice with a targeted disruption of the gene results in relatively mild retinal dysfunction, suggesting that significant therapeutic benefit may be derived even from the suppression-only component of an RNAi-based gene therapy. We show that AAV-mediated co-expression in the murine retina of a mutant human IMPDH1 gene together with short hairpin RNAs (shRNA) validated in vitro and in vivo, targeting both human and mouse IMPDH1, substantially suppresses the negative pathological effects of mutant IMPDH1, at a point where, in the absence of shRNA, expression of mutant protein in the RP10 model essentially ablates all photoreceptors in transfected areas of the retina. These data strongly suggest that an RNAi-mediated approach to therapy for RP10 holds considerable promise for human subjects.

The purpose was to evaluate MR colonography (MRC) with barium fecal tagging in detecting colorectal pathology and to determine how air-based and water-based colonic distension influences MRC. We studied 83 patients with high risk of colonic neoplasms. All received oral barium sulfate for colonic preparation before unenhanced and enhanced T1-weighted gradient-echo MRC using either water (n=54) or air (n=29) for colonic distension. Fecal tagging, distension, and artifacts were recorded. All patients underwent conventional colonoscopy within 2 weeks of MRC; the techniques were compared for detection of malignant neoplasms and polypsor=1 cm, 6-9 mm, andor=5 mm. Fecal tagging was "good" in 76% of the colonic segments in water-distended patients and 46% of air-distended patients. The degree of distension was "good" in 90.7% of water-distended patients and 44% of air-distended patients. Severe artifacts were present in 15% air-distended patients and 0.3% of water-distended patients. Both water-distended and air-distended MRC detected all malignant neoplasms and polypsor=1 cm, but more air-distended MRC were excluded for poor quality. MRC with fecal tagging is useful for detecting lesionsor=1 cm. Air distension was inferior to water distension in most aspects. Water-based colonic distension should be used for barium-tagging MRC.

DMD and BMD are X-linked myopathy diseases in most cases caused by intragenic deletions, but duplications also appear in a significant number of cases. We present a complex duplication pattern detected by MLPA, a recently formulated method applied here to amplify the 79 exons of the DMD gene. We found a double-duplication in two DMD-affected brothers and in their carrier mother, which consist of two non-contiguous duplications encompassing exons 2 to 7 and exons 50 to 55. Different models are presented to explain formation of this genetic variant.

Resumen El embarazo cervical es una infrecuente modalidad de gestacion ectopica con alto riesgo hemorragico. Su diagnostico temprano es un importante factor en el pronostico vital y reproductivo de las pacientes que presentan tal complicacion. Presentamos el caso de una paciente diagnosticada de embarazo ectopico cervical en su septima semana de gestacion. Su abordaje terapeutico fue conservador y requirio de la combinacion de metotrexato sistemico y en inyeccion intraamniotica. La menor morbimortalidad asociada y su capacidad para mantener la fertilidad hacen del tratamiento conservador una opcion terapeutica viable en casos de pacientes como el que presentamos.

This study prospectively evaluates the accuracy of contrast-enhanced ultrasound (CEUS) and dynamic magnetic resonance imaging (MRI) for the diagnosis of nodules 20 mm or smaller detected during ultrasound (US) surveillance. We included 89 patients with cirrhosis [median age, 65 years; male 53, hepatitis C virus 68, Child-Pugh A 80] without prior hepatocellular carcinoma (HCC) in whom US detected a small solitary nodule (mean diameter, 14 mm). Hepatic MRI, CEUS, and fine-needle biopsy (gold standard) (FNB) were performed at baseline. Non-HCC cases were followed (median 23 months) by CEUS/3 months and MRI/6 months. FNB was repeated up to 3 times and on detection of change in aspect/size. Intense arterial contrast uptake followed by washout in the delayed/venous phase was registered as conclusive for HCC. Final diagnoses were: HCC (n = 60), cholangiocarcinoma (n = 1), and benign lesions (regenerative/dysplastic nodule, hemangioma, focal nodular hyperplasia) (n = 28). Sex, cirrhosis cause, liver function, and alpha-fetoprotein (AFP) levels were similar between HCC and non-HCC groups. HCC patients were older and their nodules significantly larger (P < 0.0001). First biopsy was positive in 42 of 60 HCC patients. Sensitivity, specificity, and positive and negative predictive values of conclusive profile were 61.7%, 96.6%, 97.4%, and 54.9%, for MRI, 51.7%, 93.1%, 93.9%, and 50.9%, for CEUS. Values for coincidental conclusive findings in both techniques were 33.3%, 100%, 100%, and 42%. Thus, diagnosis of HCC 20 mm or smaller can be established without a positive biopsy if both CEUS and MRI are conclusive. However, sensitivity of these noninvasive criteria is 33% and, as occurs with biopsy, absence of a conclusive pattern does not rule out malignancy. These results validate the American Association for the Study of Liver Disease (AASLD) guidelines.

Purpose Mutations in the peripherin/retinal degeneration slow (RDS) gene are a known cause of various types of central retinal dystrophies. The purpose of this study was to determine the prevalence of mutations in the peripherin/RDS gene in Spanish patients with different types of autosomal dominant macular dystrophy. Methods Ophthalmic and electrophysiological examination was performed in patients from 61 unrelated autosomal dominant macular dystrophy (adMD) Spanish families. Screening for mutations in the peripherin/RDS gene by denaturing gradient gel electrophoresis (DGGE) and direct genomic sequencing was performed in index patients and extended to the family when positive. Results We report four novel mutations in peripherin/RDS and a relatively high frequency (23%) of mutations in this gene in families with adMD. Thirteen different mutations were found in fifteen adMD families. Three novel missense, four nonsense and a cis-acting splicing mutation IVS2+2T>C, were found in a Spanish population while five more missense mutations were also reported in other populations. The Arg142Trp and Arg172Trp mutations, present in several populations, were both detected in two independent Spanish families. All the missense mutations produce an amino acid substitution in the second intradiscal loop of the peripherin, while the nonsense mutations presumably generate a truncated protein. Conclusions A high frequency (23%) of mutations in the peripherin/RDS gene was found in a cohort of 61 unrelated patients with various types of autosomal dominant central retinal dystrophies as compared with a low prevalence (1.3%) of mutations in this gene causing retinitis pigmentosa in a Spanish population. Different macular dystrophy phenotypes according to the mutations in peripherin/RDS are shown. However, a limited phenotype variation was observed for these mutations within the family.

Background: Developmental eye anomalies, which include anophthalmia (absent eye) or microphthalmia (small eye) are an important cause of severe visual impairment in infants and young children. Heterozygous mutations in SOX2, a SOX1B-HMG box transcription factor, have been found in up to 10% of individuals with severe microphthalmia or anophthalmia and such mutations could also be associated with a range of non-ocular abnormalities. Methods: We performed mutation analysis on a new cohort of 120 patients with congenital eye abnormalities, mainly anophthalmia, microphthalmia and coloboma. Multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridisation (FISH) were used to detect whole gene deletion. Results: We identified four novel intragenic SOX2 mutations (one single base deletion, one single base duplication and two point mutations generating premature translational termination codons) and two further cases with the previously reported c.70del20 mutation. Of 52 patients with severe microphthalmia or anophthalmia analysed by MLPA, 5 were found to be deleted for the whole SOX2 gene and 1 had a partial deletion. In two of these, FISH studies identified sub-microscopic deletions involving a minimum of 328 Kb and 550 Kb. The SOX2 phenotypes include a patient with anophthalmia, oesophageal abnormalities and horseshoe kidney, and a patient with a retinal dystrophy implicating SOX2 in retinal development. Conclusion: Our results provide further evidence that SOX2 haploinsufficiency is a common cause of severe developmental ocular malformations and that background genetic variation determines the varying phenotypes. Given the high incidence of whole gene deletion we recommend that all patients with severe microphthalmia or anophthalmia, including unilateral cases be screened by MLPA and FISH for SOX2 deletions.

Background/Aims This study assesses the safety, pharmacokinetics and efficacy of transarterial chemoembolization using drug eluting beads (DEB), an embolizing device that slowly releases chemotherapy to decrease systemic toxicity. Methods Twenty-seven Child-Pugh A cirrhotics (76% male, 59% HCV) with untreated large/multifocal HCC received chemoembolization with doxorubicin loaded DEBs at doses adjusted for bilirubin and body surface (range: 47–150mg). Clinical and analytical data were recorded at 24 and 48h, 7, 14 and 30days after first and second TACE. Response rate was assessed by CT at 6months. Blood samples were obtained in 13 patients at 5, 20, 40, 60, 120min, 6, 24, 48 and 168h to determine doxorubicin C max and AUC. Results DEB-TACE was well tolerated with an acceptable safety profile. Two cases developed liver abscess, one leading to death. Response rate was 75% (66.6% on intention-to-treat). Doxorubicin C max and AUC were significantly lower in DEB-TACE patients (78.97±38.3ng/mL and 662.6±417.6ng/mLmin) than in conventional TACE (2341.5±3951.9ng/mL and 1812.2±1093.7ng/mLmin, p =0.00002 and p =0.001, respectively). After a median follow-up of 27.6months, 1- and 2-year survival is 92.5% and 88.9%, respectively. Conclusions Chemoembolization using DEBs is an effective procedure with a favorable pharmacokinetic profile.

We report a 22-year-old female with a variant of the Axenfeld-Rieger Syndrome (ARS) and discuss its relation with the subtelomeric 6p deletion. An ARS variant has been described in two familial cases of Axenfeld-Rieger Anomaly (ARA) featuring specific extra ocular manifestations-hypertelorism, midface hypoplasia, mild sensorial deafness, hydrocephaly, psychomotor delay and flattened femoral epiphyses. We proposed that this set of characteristics represents a separate syndrome within the ARS. On the other hand, there have been reported four cases with cryptic de novo pure 6pter microdeletions detected by specific subtelomeric probes in patients with ARS characteristics. We describe a 6pter deletion detected by SNP genotyping and confirmed by FISH and MLPA involving the FOXC1 gene in a patient with ocular and systemic findings that fit perfectly with the variant mentioned above. We conclude that the ARS variant belongs to the ARS phenotypic spectrum, which includes flattened femoral epiphyses as a feature.

Patients with relapsed/refractory chronic lymphocytic leukaemia may benefit from inclusion in clinical trials irrespective of the therapy received: a case-control retrospective analsysis