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).

Espectro mutacional de la distrofia muscular de Duchenne en España: estudio de 284 casos / Mutational spectrum of Duchenne muscular dystrophy in Spain: Study of 284 cases

Introducción: La distrofia muscular de Duchenne (DMD) es una enfermedad neuromuscular grave que afecta a uno de cada 3.500 varones nacidos y sigue un patrón de herencia ligada al cromosoma X. En esta enfermedad se observa una ausencia total de la distrofina, generalmente debida a mutaciones en el gen DMD, que altera la pauta de lectura y en torno al 80% de los casos son debidos a deleciones y duplicaciones de uno o más exones. Métodos: Se han revisado 284 casos de varones diagnosticados genéticamente de DMD entre los años 2007 y 2014. Estos pacientes provienen de 8 hospitales españoles de referencia que cubren la mayor parte del territorio español. Para la identificación de las mutaciones se realizaron las técnicas de reacción en cadena de la polimerasa multiplex, MLPA y secuenciación. Resultados: Los pacientes con DMD presentan en su mayoría grandes deleciones (46,1%) o grandes duplicaciones (19,7%) en el gen de la distrofina. El restante 34,2% corresponde al conjunto de mutaciones puntuales, destacando las sustituciones nucleotídicas tipo nonsense que aparecen en la mitad de los casos. Este estudio permitió identificar 23 nuevas mutaciones en DMD: 7 grandes deleciones y 16 mutaciones puntuales. Conclusiones: El algoritmo de diagnóstico genético aplicado por los centros participantes es el más adecuado para genotipificar a los pacientes con DMD. La especificidad genética de las distintas terapias en desarrollo pone de manifiesto la importancia de conocer la mutación de cada paciente, siendo un 38,7% de ellos susceptibles de participar en los ensayos clínicos actuales (AU)

Introduction: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive neuromuscular disease that affects one in 3500 live-born males. The total absence of dystrophin observed in DMD patients is generally caused by mutations that disrupt the reading frame of the DMD gene, and about 80% of cases harbour deletions or duplications of one or more exons. Methods: We reviewed 284 cases of males with a genetic diagnosis of DMD between 2007 and 2014. These patients were selected from 8 Spanish reference hospitals representing most areas of Spain. Multiplex PCR, MLPA, and sequencing were performed to identify mutations. Results: Most of these DMD patients present large deletions (46.1%) or large duplications (19.7%) in the dystrophin gene. The remaining 34.2% correspond to point mutations, and half of these correspond to nonsense mutations. In this study we identified 23 new mutations in DMD: 7 large deletions and 16 point mutations. Conclusions: The algorithm for genetic diagnosis applied by the participating centres is the most appropriate for genotyping patients with DMD. The genetic specificity of different therapies currently being developed emphasises the importance of identifying the mutation appearing in each patient; 38.7% of the cases in this series are eligible to participate in current clinical trials (AU)

Mutational spectrum of Duchenne muscular dystrophy in Spain: Study of 284 cases. / Espectro mutacional de la distrofia muscular de Duchenne en España: estudio de 284 casos.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive neuromuscular disease that affects one in 3500 live-born males. The total absence of dystrophin observed in DMD patients is generally caused by mutations that disrupt the reading frame of the DMD gene, and about 80% of cases harbour deletions or duplications of one or more exons. METHODS: We reviewed 284 cases of males with a genetic diagnosis of DMD between 2007 and 2014. These patients were selected from 8 Spanish reference hospitals representing most areas of Spain. Multiplex PCR, MLPA, and sequencing were performed to identify mutations. RESULTS: Most of these DMD patients present large deletions (46.1%) or large duplications (19.7%) in the dystrophin gene. The remaining 34.2% correspond to point mutations, and half of these correspond to nonsense mutations. In this study we identified 23 new mutations in DMD: 7 large deletions and 16 point mutations. CONCLUSIONS: The algorithm for genetic diagnosis applied by the participating centres is the most appropriate for genotyping patients with DMD. The genetic specificity of different therapies currently being developed emphasises the importance of identifying the mutation appearing in each patient; 38.7% of the cases in this series are eligible to participate in current clinical trials.

Nine-year experience in Gaucher disease diagnosis at the Spanish reference center Fundación Jiménez Díaz.

BACKGROUND: Fundación Jiménez Díaz (FJD) is a reference center for genetic diagnosis of Gaucher disease (GD) in Spain. Genetic analyses of acid ß-glucosidase (GBA) gene using different techniques were performed to search for new mutations, in addition to those previously and most frequently found in the Spanish population. Additionally, the study of the chitotriosidase (CHIT1) gene was used to assess the inflammatory status of patients in the follow-up of enzyme replacement therapy (ERT). We present the genetic data gathered during the last nine years at FJD. METHODS: Blood samples from patients with suspected GD were collected for enzymatic and genetic analyses. The genetic analysis was performed on DNA from 124 unrelated suspected cases and 57 relatives from 2007 to 2015, starting with a mutational screening kit, followed by Sanger sequencing of the entire gene and other techniques to look for deletions. CHIT1 was also studied to assess the reliability of this biomarker. RESULTS: In 46 out of 93 GD patients (49.5%) the two mutant alleles were found. We detected 21 different mutations. The most common mutation was N370S (c.126A > G; p.Asp409Ser current nomenclature) (in 50.5% of patients), followed by L444P (c.1448T > C; p.Leu483Pro current nomenclature) (in 24.7%). The most common heterozygous compound genotype observed (18.3%) was c.1226A > G/c.1448T > C (N370S/L444P). Two novel mutations were found (del. Ex.4-11 and c.1296G > T; pW432C), as well as p.S146L, only once previously reported. Two patients showed the homozygous state for the duplication of CHIT1. CONCLUSION: N370S and L444P are the most common mutations and other mutations associated to Parkinson's disease have been observed. This should be taken into account in the genetic counseling of GD patients.

Prenatal diagnosis of Huntington disease in maternal plasma: direct and indirect study.

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.

Two non-contiguous duplications in the DMD gene in a Spanish family.

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.

[Quick diagnosis of Prader-Willi and Angelman syndromes by means of methylation test by PCR]. / Diagnóstico rápido del síndrome de Prader-Willi y de Angelman mediante test de metilación por PCR.

OBJECTIVE: Angelman (AS) and Prader-Willi (PWS) syndromes are two different neurogenetic diseases caused by a deficiency of maternal (AS) or paternal (PWS) contributions of the region 15q11-13. Molecular diagnosis of these pathologies can be accomplished by several techniques: DNA polymorphism (microsatellite) analysis, cytogenetic techniques of fluorescent in situ hybridization (FISH) and methylation test by Southern blot (SB), with the latter being the most reliable. Recently, a new technique, based on the study of methylation through treatment with sodium bisulphite and subsequent polymerase chain reaction (PCR), has become available. We have evaluated this technique, comparing the results with those previously obtained by SB in a group of patients suspected of having PWS or AS. PATIENTS AND METHODS: Genomic DNA from 70 patients with suspected PWS or AS was used. Methylation testing by SB was carried out using the probe PW71B labeled with radioactivity. For methylation testing by PCR the DNA was treated with sodium bisulphite and hydroquinone and PCR preformed using specific primers for the maternal and paternal alleles. RESULTS: Of the 70 patients studied by PCR, 45 were normal, 17 and 8 showed altered molecular patterns that were compatible with PWS and AS, respectively. The concordance with the results obtained previously with SB was 100%. CONCLUSIONS: These data suggest that the reliability of this new technique is very good and it has advantages compared to SB, since it requires a smaller quantity of DNA and can be applied for diagnosis in newborns.