Five years' experience of the clinical exome sequencing in a Spanish single center.

Nowadays, exome sequencing is a robust and cost-efficient genetic diagnostic tool already implemented in many clinical laboratories. Despite it has undoubtedly improved our diagnostic capacity and has allowed the discovery of many new Mendelian-disease genes, it only provides a molecular diagnosis in up to 25-30% of cases. Here, we comprehensively evaluate the results of a large sample set of 4974 clinical exomes performed in our laboratory over a period of 5 years, showing a global diagnostic rate of 24.62% (1391/4974). For the evaluation we establish different groups of diseases and demonstrate how the diagnostic rate is not only dependent on the analyzed group of diseases (43.12% in ophthalmological cases vs 16.61% in neurological cases) but on the specific disorder (47.49% in retinal dystrophies vs 24.02% in optic atrophy; 18.88% in neuropathies/paraparesias vs 11.43% in dementias). We also detail the most frequent mutated genes within each group of disorders and discuss, on our experience, further investigations and directions needed for the benefit of patients.

Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes.

Despite the improved accuracy of next-generation sequencing (NGS), it is widely accepted that variants need to be validated using Sanger sequencing before reporting. Validation of all NGS variants considerably increases the turnaround time and costs of clinical diagnosis. We comprehensively assessed this need in 1109 variants from 825 clinical exomes, the largest sample set to date assessed using Illumina chemistry reported. With a concordance of 100%, we conclude that Sanger sequencing can be very useful as an internal quality control, but not so much as a verification method for high-quality single-nucleotide and small insertion/deletions variants. Laboratories might validate and establish their own thresholds before discontinuing Sanger confirmation studies. We also expand and validate 23 copy number variations detected by exome sequencing in 20 samples, observing a concordance of 95.65% (22/23).

Association of common genetic variants with risperidone adverse events in a Spanish schizophrenic population.

Risperidone non-compliance is often high due to undesirable side effects, whose development is in part genetically determined. Studies with genetic variants involved in the pharmacokinetics and pharmacodynamics of risperidone have yielded inconsistent results. Thus, the aim of this study was to investigate the putative association of genetic markers with the occurrence of four frequently observed adverse events secondary to risperidone treatment: sleepiness, weight gain, extrapyramidal symptoms and sexual adverse events. A series of 111 schizophrenia inpatients were genotyped for genetic variants previously associated with or potentially involved in risperidone response. Presence of adverse events was the main variable and potential confounding factors were considered. Allele 16Gly of ADRB2 was significantly associated with a higher risk of sexual adverse events. There were other non-significant trends for DRD3 9Gly and SLC6A4 S alleles. Our results, although preliminary, provide new candidate variants of potential use in risperidone safety prediction.

Frequency of ABCA4 mutations in 278 Spanish controls: an insight into the prevalence of autosomal recessive Stargardt disease.

AIM: To determine the carrier frequency of ABCA4 mutations in order to achieve an insight into the prevalence of autosomal recessive Stargardt disease (arSTGD) in the Spanish population. METHODS: arSTGD patients (n = 133) were analysed using ABCR400 microarray and sequencing. Control subjects were analysed by two different strategies: 200 individuals were screened for the p.Arg1129Leu mutation by denaturing-HPLC and sequencing; 78 individuals were tested for variants with the microarray and sequencing. RESULTS: For the first strategy in control subjects, the p.Arg1129Leu variant was found in two heterozygous individuals, which would mean a carrier frequency for any variant of approximately 6.0% and a calculated arSTGD prevalence of 1:1000. For the second 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. This situation should be regarded with special care when genetic counselling is given and further follow-up of these patients should be recommended.

Molecular analysis of the ABCA4 gene for reliable detection of allelic variations in Spanish patients: identification of 21 novel variants.

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.