Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing.

BACKGROUND: Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS). METHODS: Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies). RESULTS: We identified causative mutations in 17 out of the 40 patients (43%). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes. CONCLUSIONS: Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.

Post-mortem diagnosis, of cytomegalovirus and varicella zoster virus co-infection by combined histology and tissue molecular biology, in a sudden unexplained infant death.

BACKGROUND: An autopsy case of a two-month-old male infant who suddenly and unexpectedly died during his sleep, eight days after the onset of benign varicella. OBJECTIVES: To describe post-mortem combined histological and tissue molecular biological techniques for the diagnosis of cytomegalovirus and varicella zoster virus co-infection as a cause of death. STUDY DESIGN: Real-time quantitative PCR and RT-PCR assays for Herpesviruses, respiratory viruses, Adenovirus, Enterovirus and Parvovirus B19 were performed on multi-organ frozen samples and paraffin-embedded tissues in combination with histology. RESULTS: Cytomegalovirus and varicella zoster virus were detected by molecular biology with highest viral loads detected in the lungs (4.6×10(7) and 1.9×10(5) genome copies per million of cells, respectively). Pulmonary extensive necrotizing inflammation and immunohistochemistry correlated to virological data. Virological molecular biology was negative on paraffin-embedded tissues. CONCLUSIONS: This case shows that thorough quantitative virological investigations on frozen tissues must be performed in combination with histology and immunohistochemistry for the determination of the cause of a sudden unexplained infant death.

[Investigation of the sudden infant death syndrome: a multidisciplinary approach is required]. / Exploration d'une mort inattendue du nourrisson : nécessité d'une approche multidisciplinaire.

The concept of sudden infant death syndrome (SIDS) is defined as the sudden, unexpected death of an infant less than a year old which remains unexplained after in-depth investigations comprising a complete autopsy, biological analyses, and a clinical examination of the circumstances surrounding the death. This definition underlines the importance of finding the cause of this disease in order to improve preventative measures to reduce the number of deaths due to sudden infant death syndrome. Among the causes of SIDS, pediatric infectious diseases may be neglected and must be systematically sought after. We report upon a SIDS death case of a four and a half month-old that occurred during his sleep. Following the absence of an evident cause of death a scientific autopsy was performed. The histological examination of pulmonary tissue revealed broncolitic lesions associated with numerous micro-abscesses. The post mortem microbiological analyses revealed evidence of an infection by the respiratory syncytial virus complicated by a bacterial infection due to Haemophilus influenzae. The case underlines the necessity of a multidisciplinary approach to researching SIDS, involving both clinicians and biologists, in order to determine the causes of these deaths.