From the reward network to whole-brain metrics: structural connectivity in adolescents and young adults according to body mass index and genetic risk of obesity.

BACKGROUND: Obesity is a multifactorial condition. Genetic variants, such as the fat mass and obesity related gene (FTO) polymorphism, may increase the vulnerability of developing obesity by disrupting dopamine signaling within the reward network. Yet, the association of obesity, genetic risk of obesity, and structural connectivity of the reward network in adolescents and young adults remains unexplored. We investigate, in adolescents and young adults, the structural connectivity differences in the reward network and at the whole-brain level according to body mass index (BMI) and the FTO rs9939609 polymorphism. METHODS: One hundred thirty-two adolescents and young adults (age range: [10, 21] years, BMI z-score range: [-1.76, 2.69]) were included. Genetic risk of obesity was determined by the presence of the FTO A allele. Whole-brain and reward network structural connectivity were analyzed using graph metrics. Hierarchical linear regression was applied to test the association between BMI-z, genetic risk of obesity, and structural connectivity. RESULTS: Higher BMI-z was associated with higher (B = 0.76, 95% CI = [0.30, 1.21], P = 0.0015) and lower (B = -0.003, 95% CI = [-0.006, -0.00005], P = 0.048) connectivity strength for fractional anisotropy at the whole-brain level and of the reward network, respectively. The FTO polymorphism was not associated with structural connectivity nor with BMI-z. CONCLUSIONS: We provide evidence that, in healthy adolescents and young adults, higher BMI-z is associated with higher connectivity at the whole-brain level and lower connectivity of the reward network. We did not find the FTO polymorphism to correlate with structural connectivity. Future longitudinal studies with larger sample sizes are needed to assess how genetic determinants of obesity change brain structural connectivity and behavior.

Effect of the catechol-O-methyltransferase Val 158 Met polymorphism on theory of mind in obesity.

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.

New COL6A6 variant detected by whole-exome sequencing is linked to break points in intron 4 and 3'-UTR, deleting exon 5 of RHO, and causing adRP.

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.

The interaction effect between BDNF val66met polymorphism and obesity on executive functions and frontal structure.

The prevalence of obesity is increasing worldwide. Previous research has shown a relationship between obesity and both executive functioning alterations and frontal cortex volume reductions. The Brain Derived Neurotrophic Factor val66met polymorphism, involved in eating behavior, has also been associated with executive functions and prefrontal cortex volume, but to date it has not been studied in relation to obesity. Our aim is to elucidate whether the interaction between the Brain Derived Neurotrophic Factor val66met polymorphism and obesity status influences executive performance and frontal-subcortical brain structure. Sixty-one volunteers, 34 obese and 27 controls, age range 12-40, participated in the study. Participants were assigned to one of two genotype groups (met allele carriers, n = 16, or non-carriers, n = 45). Neuropsychological assessment comprised the Trail Making Test, the Stroop Test and the Wisconsin Card Sorting Test, all tasks that require response inhibition and cognitive flexibility. Subjects underwent magnetic resonance imaging in a Siemens TIM TRIO 3T scanner and images were analyzed using the FreeSurfer software. Analyses of covariance controlling for age and intelligence showed an effect of the obesity-by-genotype interaction on perseverative responses on the Wisconsin Card Sorting Test as well as on precentral and caudal middle frontal cortical thickness: obese met allele carriers showed more perseverations on the Wisconsin Card Sorting Test and lower frontal thickness than obese non-carriers and controls. In conclusion, the Brain Derived Neurotrophic Factor may play an important role in executive functioning and frontal brain structure in obesity.

Next-generation sequencing-based gene panel tests for the detection of rare variants and hypomorphic alleles associated with primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is a complex disease with a strong hereditably component. Several genetic variants have recently been associated with POAG, partially due to technological improvements such as next-generation sequencing (NGS). The aim of this study was to genetically analyze patients with POAG to determine the contribution of rare variants and hypomorphic alleles associated with glaucoma as a future method of diagnosis and early treatment. Seventy-two genes potentially associated with adult glaucoma were studied in 61 patients with POAG. Additionally, we sequenced the coding sequence of CYP1B1 gene in 13 independent patients to deep analyze the potential association of hypomorphic CYP1B1 alleles in the pathogenesis of POAG. We detected nine rare variants in 16% of POAG patients studied by NGS. Those rare variants are located in CYP1B1, SIX6, CARD10, MFN1, OPTC, OPTN, and WDR36 glaucoma-related genes. Hypomorphic variants in CYP1B1 and SIX6 genes have been identified in 8% of the total POAG patient assessed. Our findings suggest that NGS could be a valuable tool to clarify the impact of genetic component on adult glaucoma. However, in order to demonstrate the contribution of these rare variants and hypomorphic alleles to glaucoma, segregation and functional studies would be necessary. The identification of new variants and hypomorphic alleles in glaucoma patients will help to configure the genetic identity of these patients, in order to make an early and precise molecular diagnosis.

SpadaHC: a database to improve the classification of variants in hereditary cancer genes in the Spanish population.

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/.

Detection of novel mutations that cause autosomal dominant retinitis pigmentosa in candidate genes by long-range PCR amplification and next-generation sequencing.

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.

Survey of familial glaucoma shows a high incidence of cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) mutations in non-consanguineous congenital forms in a Spanish population.

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.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing.

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.

Mutant PRPF8 Causes Widespread Splicing Changes in Spliceosome Components in Retinitis Pigmentosa Patient iPSC-Derived RPE Cells.

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.

Transcriptional expression of cis-acting and trans-acting splicing mutations cause autosomal dominant retinitis pigmentosa.

Two types of mutations may lead to deficient pre-mRNA splicing: cis-acting mutations that inactivate a constitutive or alternative splice site within the pre-mRNA, and trans-acting mutations that affect the function of a basal factor of the splicing machinery. Autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in at least 12 genes, with mutations in rhodopsin being the most prevalent. Two cis-acting mutations, g.3811A>G and g.5167G>T at the splice site in the rhodopsin gene (RHO; GenBank U49742.1) are linked to adRP in a Spanish population; while a cis-acting mutation, g.4335G>T, has been linked to recessive RP (arRP). Transcriptional expression analysis showed that the cis-acting splicing mutations linked to adRP promoted alternative splice sites, while the arRP linked mutation results in exclusion of exon 4. Trans-acting splicing mutations associated with adRP have also been found, and mutations in the pre-mRNA splicing factors PRPF3, PRPF8, PRPF31, and RP9 are associated with adRP in several populations. This report describes a new mutation in PRPF3 in a Spanish adRP family. We also investigated the transcriptional patterns in Epstein-Barr virus (EBV)-transformed lymphoblastoid cells from patients carrying a mutation in PRPF8. Despite the role of PRPF8 in the minor U12 splicing processes, microarray analysis revealed that mutations in PRPF8 not only did not result in significant differences in splicing efficiency of rhodopsin, but no apparent changes in expression of U12-type intron genes and splicing processes was observed. Microarray analysis revealed a panel of differentially expressed genes mapped to the RP loci, and future work will determine their role in RP.

Novel LMX1B mutation in familial nail-patella syndrome with variable expression of open angle glaucoma.

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.

High prevalence of mutations in peripherin/RDS in autosomal dominant macular dystrophies in a Spanish population.

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.

Generation of a human iPSC line from a patient with retinitis pigmentosa caused by mutation in PRPF8 gene.

The human iPSC cell line, RP2-FiPS4F1 (RCPFi001-A), derived from dermal fibroblasts from the patient with retinitis pigmentosa caused by the mutation of the gene PRPF8, was generated by non-integrative reprogramming technology using OCT3/4, SOX2, CMYC and KLF4 reprogramming factors.

Three novel and the common Arg677Ter RP1 protein truncating mutations causing autosomal dominant retinitis pigmentosa in a Spanish population.

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.

Sequence variations in the retinal fascin FSCN2 gene in a Spanish population with autosomal dominant retinitis pigmentosa or macular degeneration.

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 pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 in Spanish families with autosomal dominant retinitis pigmentosa.

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.

Detection of genomic variations in BRCA1 and BRCA2 genes by long-range PCR and next-generation sequencing.

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.

High-resolution melting analysis of the common c.1905+1G>A mutation causing dihydropyrimidine dehydrogenase deficiency and lethal 5-fluorouracil toxicity.

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.