Implementation of a Reliable Next-Generation Sequencing Strategy for Molecular Diagnosis of Dystrophinopathies

International audienceDiagnosis of dystrophinopathies needs to combine several techniques for detecting copy number variations (CNVs; two-thirds of mutations) and single nucleotide variations (SNVs). We participated in the design of an amplicon-based PCR kit (Multiplicom) for sequencing with a GS-Junior instrument (Roche) and later with a MiSeq instrument (Illumina). We compared two different software programs, MiSeq Reporter (Illumina) and SeqNext (JSI Medical Systems) for data analyses. Testing of six patient DNA samples carrying 72 SNVs in the DMD gene showed an experimental sensitivity of 91.7% with MiSeq Reporter, 98.6% with SeqNext, and >99.9% with both, demonstrating the need to use two different software programs. Analytical specificity was >98%. Fifty-eight additional patient DNAs were analyzed, and 25 deleterious mutations were identified, without false-negative results. We also tested the possibility for our protocol to identify CNVs. We performed additional next-generation sequencing experiments on 50 DNAs and identified 28 CNVs, all confirmed by multiple ligation probe amplification. Statistical analyses on amplicons without CNV (n = 3797), amplicons with heterozygous deletions (n = 51) or duplications (n = 191), and with hemizygous duplications (n = 63) showed a sensitivity and specificity of >99.9%. We implemented a strategy to simultaneously detect SNVs and CNVs in the DMD gene with one comprehensive technique, allowing considerable reduction of time and cost burden for diagnosis of dystrophinopathies

Brief report: 11p15 imprinting center region 1 loss of methylation is a common and specific cause of typical Russell-Silver syndrome: Clinical scoring system and epigenetic-phenotypic correlations

Context: Russell-Silver syndrome (RSS), characterized by intrauterine and postnatal growth retardation, dysmorphic features, and frequent body asymmetry, spares cranial growth. Maternal uniparental disomy for chromosome 7 (mUPD7) is found in 5-10% of cases. We identified loss of methylation (LOM) of 11p15 Imprinting Center Region 1 (ICR1) domain (including IGF-II) as a mechanism leading to RSS. Objective: The aim was to screen for 11p15 epimutation and mUPD7 in RSS and non-RSS small-for-gestational-age (SGA) patients and identify epigenetic-phenotypic correlations. Studied Population and Methods: A total of 127 SGA patients were analyzed. Clinical diagnosis of RSS was established when the criterion of being SGA was associated with at least three of five criteria: postnatal growth retardation, relative macrocephaly, prominent forehead, body asymmetry, and feeding difficulties. Serum IGF-II was evaluated for 82 patients. Results: Of the 127 SGA patients, 58 were diagnosed with RSS; 37 of these (63.8%) displayed partial LOM of the 11p15 ICR1 domain, and three (5.2%) had mUPD7. No molecular abnormalities were found in the non-RSS SGA group (n = 69). Birth weight, birth length, and postnatal body mass index (BMI) were lower in the abnormal 11p15 RSS group (ab-ICR1-RSS) than in the normal 11p15 RSS group [-3.4 vs. -2.6 SD score (SDS), -4.4 vs. -3.4 SDS, and -2.5 vs. -1.6 SDS, respectively; P < 0.05]. Among RSS patients, prominent forehead, relative macrocephaly, body asymmetry, and low BMI were significantly associated with ICR1 LOM. All ab-ICR1-RSS patients had at least four of five criteria of the scoring system. Postnatal IGF-II levels were within normal values. Conclusion: The 11p15 ICR1 epimutation is a major, specific cause of RSS exhibiting failure to thrive. We propose a clinical scoring system (including a BMI < -2 SDS), highly predictive of 11p15 ICR1 LOM, for the diagnosis of RSS. Copyright © 2007 by The Endocrine Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe