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Abstract Background Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. Methods We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. Results We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. Conclusions HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Abstract Background Retinitis pigmentosa (RP), a clinically and genetically heterogeneous group of retinal degeneration disorders affecting the photoreceptor cells, is one of the leading causes of genetic blindness. Mutations in the photoreceptor-specific gene RP1 account for 3–10% of cases of autosomal dominant RP (adRP). Most of these mutations are clustered in a 500 bp region of exon 4 of RP1. Methods Denaturing gradient gel electrophoresis (DGGE) analysis and direct genomic sequencing were used to evaluate the 5' coding region of exon 4 of the RP1 gene for mutations in 150 unrelated index adRP patients. Ophthalmic and electrophysiological examination of RP patients and relatives according to pre-existing protocols were carried out. Results Three novel disease-causing mutations in RP1 were detected: Q686X, K705fsX712 and K722fsX737, predicting truncated proteins. One novel missense mutation, Thr752Met, was detected in one family but the mutation does not co-segregate in the family, thereby excluding this amino acid variation in the protein as a cause of the disease. We found the Arg677Ter mutation, previously reported in other populations, in two independent families, confirming that this mutation is also present in a Spanish population. Conclusion Most of the mutations reported in the RP1 gene associated with adRP are expected to encode mutant truncated proteins that are approximately one third or half of the size of wild type protein. Patients with mutations in RP1 showed mild RP with variability in phenotype severity. We also observed several cases of non-penetrant mutations.

Retinitis pigmentosa (RP) is a rare, progressive disease that affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression of or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue, and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell-type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre-mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell-type specificity through differential expression and alternative splicing (AS) and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by a specific splicing program and identifies retinal AS events as a framework toward the design of novel therapeutic opportunities. [ABSTRACT FROM AUTHOR]

Obesity is often accompanied with psychosocial adjustment problems, such as difficulties in social interactions and social withdrawal. A key aspect of social cognition is theory of mind, which allows inferring mental states, feelings, motivations, and beliefs of others and to use this information to predict their future behaviour. Theory of mind is highly dependent on prefrontal dopaminergic neurotransmission, which is regulated by catechol‐O‐methyltransferase (COMT) activity. We aimed at determining whether theory of mind is altered in obesity and if this ability is modulated by COMT. Fifty patients with obesity and 47 normal‐weight individuals underwent the Reading the Mind in the Eyes Test, the Wisconsin Card Sorting Test, and the Vocabulary subscale of the Wechsler Adult Intelligence Scale. The genotype for the COMT Val 158Met functional polymorphism was determined for all subjects. Patients with obesity obtained significantly lower scores in the negative items of the Reading the Mind in the Eyes Test than normal‐weight subjects. Further, an interaction effect was observed between group and COMT genotype. Specifically, the presence of the Met allele was associated to a better identification of negative mental states only in patients with obesity. Our results indicate that obesity is accompanied with difficulties in theory of mind and that this ability is influenced by the COMT genotype. [ABSTRACT FROM AUTHOR]

Obesity is a multifactorial disease caused by the interaction between genotype and environment, and it is considered to be a type of addictive alteration. The A1 allele of the DRD2/ANKK1-TaqIA gene has been associated with addictive disorders, with obesity and with the performance in executive functions. The 7 repeat allele of the DRD4 gene has likewise been associated with the performance in executive functions, as well as with addictive behaviors and impulsivity. Participants were included in the obesity group (N = 42) if their body mass index (BMI) was equal to or above 30, and in the lean group (N = 42) if their BMI was below 25. The DRD2/ANKK1-TaqIA and DRD4 VNTR polymorphisms were obtained. All subjects underwent neuropsychological assessment. Eating behavior traits were evaluated. The 'DRD2/ANKK1-TaqIA A1-allele status' had a significant effect on almost all the executive variables, but no significant 'DRD4 7R-allele status' effects were observed for any of the executive variables analyzed. There was a significant 'group' × 'DRD2/ANKK1-TaqIA A1-allele status' interaction effect on LN and 'group' × 'DRD4 7R-allele status' interaction effect on TMT B-A score. Being obese and a carrier of the A1 allele of DRD2/ANKK1-TaqIA or the 7R allele of DRD4 VNTR polymorphisms could confer a weakness as regards the performance of executive functions. [ABSTRACT FROM AUTHOR]

The article focuses on nail-patella syndrome with mutation in the LMX1B gene. The study of the LMX1B gene showed mutation c.728G>C in heterozygosis. In nail-patella syndrome, more than 130 different mutations have been described that consist of changes in one nucleotide. Mutation in the LMX1B gene avoids the need to perform a renal biopsy.

Accurate classification of genetic variants is crucial for clinical decision-making in hereditary cancer. In Spain, genetic diagnostic laboratories have traditionally approached this task independently due to the lack of a dedicated resource. Here we present SpadaHC, a web-based database for sharing variants in hereditary cancer genes in the Spanish population. SpadaHC is implemented using a three-tier architecture consisting of a relational database, a web tool and a bioinformatics pipeline. Contributing laboratories can share variant classifications and variants from individuals in Variant Calling Format (VCF) format. The platform supports open and restricted access, flexible dataset submissions, automatic pseudo-anonymization, VCF quality control, variant normalization and liftover between genome builds. Users can flexibly explore and search data, receive automatic discrepancy notifications and access SpadaHC population frequencies based on many criteria. In February 2024, SpadaHC included 18 laboratory members, storing 1.17 million variants from 4306 patients and 16 343 laboratory classifications. In the first analysis of the shared data, we identified 84 genetic variants with clinically relevant discrepancies in their classifications and addressed them through a three-phase resolution strategy. This work highlights the importance of data sharing to promote consistency in variant classifications among laboratories, so patients and family members can benefit from more accurate clinical management. Database URL: https://spadahc.ciberisciii.es/., (© The Author(s) 2024. Published by Oxford University Press.)

Obesity is often accompanied with psychosocial adjustment problems, such as difficulties in social interactions and social withdrawal. A key aspect of social cognition is theory of mind, which allows inferring mental states, feelings, motivations, and beliefs of others and to use this information to predict their future behaviour. Theory of mind is highly dependent on prefrontal dopaminergic neurotransmission, which is regulated by catechol-O-methyltransferase (COMT) activity. We aimed at determining whether theory of mind is altered in obesity and if this ability is modulated by COMT. Fifty patients with obesity and 47 normal-weight individuals underwent the Reading the Mind in the Eyes Test, the Wisconsin Card Sorting Test, and the Vocabulary subscale of the Wechsler Adult Intelligence Scale. The genotype for the COMT Val 158 Met functional polymorphism was determined for all subjects. Patients with obesity obtained significantly lower scores in the negative items of the Reading the Mind in the Eyes Test than normal-weight subjects. Further, an interaction effect was observed between group and COMT genotype. Specifically, the presence of the Met allele was associated to a better identification of negative mental states only in patients with obesity. Our results indicate that obesity is accompanied with difficulties in theory of mind and that this ability is influenced by the COMT genotype., (© 2019 John Wiley & Sons, Ltd and Eating Disorders Association.)

Purpose: This study aimed to test a newly devised cost-effective multiplex PCR assay for the molecular diagnosis of autosomal dominant retinitis pigmentosa (adRP), as well as the use of whole-exome sequencing (WES) to detect disease-causing mutations in adRP., Methods: Genomic DNA was extracted from peripheral blood lymphocytes of index patients with adRP and their affected and unaffected family members. We used a newly devised multiplex PCR assay capable of amplifying the genetic loci of RHO, PRPH2, RP1, PRPF3, PRPF8, PRPF31, IMPDH1, NRL, CRX, KLHL7, and NR2E3 to molecularly diagnose 18 index patients with adRP. We also performed WES in affected and unaffected members of four families with adRP in whom a disease-causing mutation was previously not found., Results: We identified five previously reported mutations (p.Arg677X in the RP1 gene, p.Asp133Val and p.Arg195Leu in the PRPH2 gene, and p.Pro171Leu and p.Pro215Leu in the RHO gene) and one novel mutation (p.Val345Gly in the RHO gene) representing 33% detection of causative mutations in our adRP cohort. Comparative WES analysis showed a new variant (p.Gly103Arg in the COL6A6 gene) that segregated with the disease in one family with adRP. As this variant was linked with the RHO locus, we sequenced the complete RHO gene, which revealed a deletion in intron 4 that encompassed all of exon 5 and 28 bp of the 3'-untranslated region (UTR)., Conclusions: The novel multiplex PCR assay with next-generation sequencing (NGS) proved effective for detecting most of the adRP-causing mutations. A WES approach led to identification of a deletion in RHO through detection of a new linked variant in COL6A6. No pathogenic variants were identified in the remaining three families. Moreover, NGS and WES were inefficient for detecting the complete deletion of exon 5 in the RHO gene in one family with adRP. Carriers of this deletion showed variable clinical status, and two of these carriers had not previously been diagnosed with RP.

Purpose: To identify myocilin (MYOC) and cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) mutations in a Spanish population with different clinical forms of familial glaucoma or ocular hypertension (OHT)., Methods: Index patients from 226 families participated in this study. Patients were diagnosed with familial glaucoma or OHT by complete ophthalmologic examination. Screening for MYOC mutations was performed in 207 index patients: 96 with adult-onset primary open-angle glaucoma (POAG), 21 with primary congenital glaucoma (PCG), 18 with juvenile-onset open-angle glaucoma (JOAG), five with Axenfeld-Rieger syndrome (ARS), and 67 with other types of glaucoma. One hundred two of the families (including all those in whom a MYOC mutation was detected) were also screened for CYP1B1 mutations: 45 POAG, 25 PCG, 21 JOAG, four ARS, and seven others., Results: We examined 292 individuals (patients and relatives) with a positive family history of glaucoma or OHT. We identified two novel MYOC variants, p.Lys39Arg and p.Glu218Lys, in two families with POAG, and six previously reported MYOC mutations in seven families with POAG (four), JOAG (one), PCG (one), and normotensive glaucoma (one). CYP1B1 mutations were found in 16 index patients with PCG (nine), POAG (three), JOAG (two), and ARS (two)., Conclusions: The high percentage (9/25=36%) of mutations in CYP1B1 found in non-consanguineous patients with congenital glaucoma mandates genetic testing. However, the percentage of mutations (9/207=4.4%) in MYOC associated with glaucoma is relatively low in our population. The variable phenotype expression of glaucoma, even in families, cannot be explained with a digenic mechanism between MYOC and CYP1B1.

Dihydropyrimidine dehydrogenase (DPD) deficiency is a pharmacogenetic syndrome associated with life-threatening toxicity following exposure to the fluoropyrimidine drugs 5-fluorouracil (5-FU) and capecitabine (CAP), widely used for the treatment of colorectal cancer and other solid tumors. The most prominent loss-of-function allele of the DPYD gene is the splice-site mutation c.1905+1G>A. In this study we report the case of a 73-year old woman with metastatic colorectal cancer who died from drug-induced toxicity after the first cycle of 5-FU-containing chemotherapy. Her symptoms included severe neutropenia, thrombocytopenia, mucositis and diarrhea; she died 16 days later despite intensive care measures. Post-mortem genetic analysis revealed that the patient was homozygous for the c.1905+1G>A deleterious allele and several family members consented to being screened for this mutation. This is the first report in Spain of a case of 5-FU-induced lethal toxicity associated with a genetic defect that results in the complete loss of the DPD enzyme. Although the frequency of c.1905+1G>A carriers in the white population ranges between 1 and 2%, the few data available for the Spanish population and the severity of this case prompted us to design a genotyping procedure to prevent future toxic effects of 5-FU/CAP. Since our group had previously developed a high-resolution melting (HRM) assay for the simultaneous detection of KRAS, BRAF, and/or EGFR somatic mutations in colorectal and lung cancer patients considered for EGFR-targeted therapies, we included the DPYD c.1905+1G>A mutation in the screening test that we describe herein. HRM provides a rapid, sensitive, and inexpensive method that can be easily implemented in diagnostic settings for the routine pre-therapeutic testing of a gene mutation panel with implications in the pharmacologic treatment.

Purpose: To devise an effective method for detecting mutations in 12 genes (CA4, CRX, IMPDH1, NR2E3, RP9, PRPF3, PRPF8, PRPF31, PRPH2, RHO, RP1, and TOPORS) commonly associated with autosomal dominant retinitis pigmentosa (adRP) that account for more than 95% of known mutations., Methods: We used long-range PCR (LR-PCR) amplification and next-generation sequencing (NGS) performed in a GS Junior 454 benchtop sequencing platform. Twenty LR-PCR fragments, between 3,000 and 10,000 bp, containing all coding exons and flanking regions of the 12 genes, were obtained from DNA samples of patients with adRP. Sequencing libraries were prepared with an enzymatic (Fragmentase technology) method., Results: Complete coverage of the coding and flanking sequences of the 12 genes assayed was obtained with NGS, with an average sequence depth of 380× (ranging from 128× to 1,077×). Five previous known mutations in the adRP genes were detected with a sequence variation percentage between 35% and 65%. We also performed a parallel sequence analysis of four samples, three of them new patients with index adRP, in which two novel mutations were detected in RHO (p.Asn73del) and PRPF31 (p.Ile109del)., Conclusions: The results demonstrate that genomic LR-PCR amplification together with NGS is an effective method for analyzing individual patient samples for mutations in a monogenic heterogeneous disease such as adRP. This approach proved effective for the parallel analysis of adRP and has been introduced as routine. Additionally, this approach could be extended to other heterogeneous genetic diseases.

Advances in sequencing technologies, such as next-generation sequencing (NGS), represent an opportunity to perform genetic testing in a clinical scenario. In this study, we developed and tested a method for the detection of mutations in the large BRCA1 and BRCA2 tumor suppressor genes, using long-range PCR (LR-PCR) and NGS, in samples from individuals with a personal and/or family history of breast and/or ovarian cancer. Eleven LR-PCR fragments, between 3000 and 15,300 bp, containing all coding exons and flanking splice junctions of BRCA1 and BRCA2, were obtained from DNA samples of five individuals carrying mutations in either BRCA1 or BRCA2. Libraries for NGS were prepared using an enzymatic (Nextera technology) method. We analyzed five individual samples in parallel by NGS and obtained complete coverage of all LR-PCR fragments, with an average coding sequence depth for each nucleotide of >30 reads, running from ×7 (in exon 22 of BRCA1) to >×150. We detected and confirmed 100% of the mutations that predispose to the risk of cancer, together with other genomic variations in BRCA1 and BRCA2. Our approach demonstrates that genomic LR-PCR, together with NGS, using the GS Junior 454 System platform, is an effective method for patient sample analysis of BRCA1 and BRCA2 genes. In addition, this method could be performed in regular molecular genetics laboratories., (Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices., Methods: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform., Results: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations., Conclusions: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Purpose: To investigate the cellular expression of cis-acting splicing mutations in the rhodopsin gene (RHO) that lead to autosomal dominant or recessive retinitis pigmentosa (adRP/arRP) and the role of nonsense-mediated mRNA decay (NMD) in its pathogenic mechanism. To design a potential therapeutic RNAi-based suppression strategy for cis-acting adRP splicing mutants., Methods: Cells were transfected with genomic constructs encoding the human wild-type (WT) and c.531-2A>G, c.936+1G>T, c.937-1G>T and c.745G>T RHO mutants. Total RNA was quantified by RT-PCR and protein was analyzed by immunocytochemistry. Three small interfering (si)RNAs directed against adRP mutant transcripts were designed and assayed in COS7 cells., Results: The RHO cis-acting splicing mutations causing adRP, c.531-2A>G and c.937-1G>T, induce cryptic splicing. In contrast, the c.936+1G>T mutation, which causes arRP, results in exon skipping. Although the c.531-2A>G and c.745G>T RHO sequence predicted a premature termination codon (PTC) that should be a target for NMD, these mutant proteins were detected in transfected cells. The siRNAs designed to interfere with adRP mutants silenced the corresponding mRNA with varying efficiency., Conclusions: Although two RHO mutations that cause different RP phenotypes were the target for the NMD mechanism, a fraction of mutant RNA transcript may circumvent the NMD mechanism and be translated into protein. Thus, different levels of mutant protein may be necessary to trigger the RP phenotype. The findings demonstrate the potential use of siRNA to interfere with cis-acting splicing RHO transcripts. However, limitations in the mutation sequence and incomplete mutant transcript elimination should be considered in a therapeutic approach for adRP.

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.

Purpose: To describe the genetic and clinical findings in a large Spanish pedigree with nail-patella syndrome (NPS) and to investigate the expressivity of open angle glaucoma (OAG) in the family members., Methods: All individuals underwent a complete ophthalmologic examination, including optical coherence tomography (OCT) of the optic disc and peripapillary region and ultrasound pachymetry. Screening for mutations in the LMX1B gene was performed by denaturing gradient gel electrophoresis and direct genomic sequencing analysis., Results: Ten family members had NPS, seven with varying degrees of ocular hypertension (OHT). Only one of these had advanced OAG. The others showed high pachymetry values and OCT retinal nerve fiber layer (RNFL) thickness above the normal values. Screening for mutations in the exonic and flanking sequences of the LMX1B gene showed a deletion of one G (289delG) within the coding sequence of exon 3 at codon 97, resulting in a frame shift that creates a premature stop at codon 105 (E97fsX105), predicting a truncated protein. This mutation was present in all NPS patients and absent in the unaffected family members., Conclusions: A novel mutation in the homeobox transcription factor LMX1B causes NPS in a family with variable expressivity of the syndrome, including OAG. The pathogenic mechanism resulting from the mutation is presumably haploinsufficiency rather than a dominant negative effect, which would explain the clinical variability in this family. All NPS OHT patients had considerably thick corneas and RNFL.

Background: Retinitis pigmentosa (RP), a clinically and genetically heterogeneous group of retinal degeneration disorders affecting the photoreceptor cells, is one of the leading causes of genetic blindness. Mutations in the photoreceptor-specific gene RP1 account for 3-10% of cases of autosomal dominant RP (adRP). Most of these mutations are clustered in a 500 bp region of exon 4 of RP1., Methods: Denaturing gradient gel electrophoresis (DGGE) analysis and direct genomic sequencing were used to evaluate the 5' coding region of exon 4 of the RP1 gene for mutations in 150 unrelated index adRP patients. Ophthalmic and electrophysiological examination of RP patients and relatives according to pre-existing protocols were carried out., Results: Three novel disease-causing mutations in RP1 were detected: Q686X, K705fsX712 and K722fsX737, predicting truncated proteins. One novel missense mutation, Thr752Met, was detected in one family but the mutation does not co-segregate in the family, thereby excluding this amino acid variation in the protein as a cause of the disease. We found the Arg677Ter mutation, previously reported in other populations, in two independent families, confirming that this mutation is also present in a Spanish population., Conclusion: Most of the mutations reported in the RP1 gene associated with adRP are expected to encode mutant truncated proteins that are approximately one third or half of the size of wild type protein. Patients with mutations in RP1 showed mild RP with variability in phenotype severity. We also observed several cases of non-penetrant mutations.

Purpose: Only one mutation in the retinal fascin gene (FSCN2) has so far been associated with autosomal dominant retinitis pigmentosa (adRP) and macular dystrophy (adMD), in a Japanese population. Our study was designed to identify mutations in the FSCN2 gene among Spanish persons with adRP or adMD., Methods: Denaturing gradient gel electrophoresis and direct genomic sequencing were used to evaluate the complete coding region and flanking intronic sequences of the FSCN2 gene for mutations in 150 unrelated adRP and 15 adMD index patients, and in 50 sporadic cases of retinitis pigmentosa, together with 50 controls. Ophthalmic and electrophysiological examination of retinitis pigmentosa patients and their relatives was carried out according to pre-existing protocols., Results: Sixteen nucleotide substitutions were detected in the coding sequence of the index patients. Nine of these, His7Tyr, Ala122Thr, Ser126Phe, His138Tyr, Arg149Gln, Ala240Thr, Ala323Thr, Asn331His, and Phe367Leu are missense mutations, one is a nonsense mutation (Lys302Stop), and six are silent mutations. Co-segregation of the mutations in the families showed no direct relation between mutation and disease., Conclusions: The photoreceptor-specific FSCN2 gene showed a relatively high number of sequence variations. The mutation 208delG in FSCN2, the only mutation so far associated with adRP or adMD, and which presumably causes a null allele, was not detected in these Spanish families. The nonsense mutation, Lys302Stop, detected in one adRP Spanish family is not the cause of the disease. These findings support the fact that the kind and frequency of the mutations depend on the ethnic population.

Mutations in the c-KIT gene have been identified in many sporadic and familial cases of gastrointestinal stromal tumor (GIST). We report a familial case of GIST with cutaneous hyperpigmentation associated with a novel germline mutation in the c-KIT gene. Screening for mutations in exon 11 of the c-KIT gene in genomic DNA from tumors and peripheral blood of the members of a family with GISTs was undertaken by direct genomic sequencing. Tumors from GIST patients were analyzed histologically and immunohistochemically. Clinical examination of GIST patients was also performed to detect other systemic diseases associated with c-KIT mutations. Histological study showed that the tumors were GISTs expressing CD34 and c-KIT protein. This GIST-hyperpigmentation disease was associated in the family with a germline mutation in the c-KIT gene. The mutation is a duplication of the sequence CAACTT located in exon 11 of the c-KIT gene, which introduces two extra glutamine and leucine residues in the encoding protein between positions 576 and 577. This Spanish family was affected with GISTs and cutaneous hyperpigmentation associated with a novel germline mutation Leu576_Pro577insGlnLeu in the juxtamembrane domain of the c-KIT receptor. These types of mutation in the c-KIT gene activate the tyrosine kinase activity of the c-KIT receptor and induce constitutive signaling leading to GISTs, in some cases associated with cutaneous hyperpigmentation., ((c) 2005 Wiley-Liss, Inc.)

Purpose: Mutations in the systemically expressed pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 have recently been associated with autosomal dominant retinitis pigmentosa (adRP). This study was intended to identify mutations in PRPF3, PRPF8, and PRPF31 in 150 Spanish families affected by adRP, to measure the contribution of mutations in these genes to adRP in that population, and to correlate RP phenotype expression with mutations in pre-mRNA splicing-factor genes., Methods: Denaturing gradient gel electrophoresis (DGGE) and direct genomic sequencing were used to evaluate the complete coding region and flanking intronic sequences of the PRPF31 gene, exon 42 of PRPF8, and exon 11 of PRPF3 for mutations in 150 unrelated index patients with adRP. Ophthalmic and electrophysiological examination of patients with RP and their relatives was performed according to preexisting protocols., Results: Three nonsense mutations caused by insertion and deletion sequences and two missense mutations (Arg2310Gly) and within the stop codon of the PRPF8 gene (TGA-->TTG), were detected in five unrelated heterozygous patients. Three patients were heterozygous carriers of different nonsense mutations in exon 8 of the PRPF31, gene and one Thr494Met mutation was found in exon 11 of the PRPF3 gene. Cosegregation of the mutation in PRPF8 and PRPF3 with adRP was observed. However, two nonsense mutations in PRPF31 causing adRP detected in two families showed asymptomatic carriers., Conclusions: Nine mutations, six of which are novel, in the pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31, causing adRP have been identified in the Spanish population. Their contribution to adRP is approximately 5% after correction in relation to mutations found in other genes causing adRP. The patients carrying a mutation in the pre-mRNA splicing-factor PRPF8 gene showed a type 1 diffuse RP. The existence of asymptomatic carriers of the nonsense mutation in the PRPF31 gene suggests incomplete penetrance for these mutations in the families.