Homozygous mutations of the telomeric SMN1 gene lead to degeneration of motor neurons causing spinal muscular atrophy (SMA). A highly similar centromeric gene (SMN2) can only partially compensate for SMN1 deficiency. The c.859G>C variant in SMN2 has been recently reported as a positive disease modifier. We identified the variant in 10 unrelated chronic SMA patients with a wide spectrum of phenotypes ranging from type II patients who can only sit to adult walkers. Haplotype analysis strongly suggests that the variant originated from a common ancestor. Our results confirm that the c.859G>C variant is a milder SMN2 allele and predict a direct correlation between SMN activity and phenotypic severity.
BackgroundSensorineural hearing loss (SNHL) is the most common sensory impairment. Comprehensive next-generation sequencing (NGS) has become the standard for the etiological diagnosis of early-onset SNHL. However, accurate selection of target genomic regions (gene panel/exome/genome), analytical performance and variant interpretation remain relevant difficulties for its clinical implementation.MethodsWe developed a novel NGS panel with 199 genes associated with non-syndromic and/or syndromic SNHL. We evaluated the analytical sensitivity and specificity of the panel on 1624 known single nucleotide variants (SNVs) and indels on a mixture of genomic DNA from 10 previously characterized lymphoblastoid cell lines, and analyzed 50 Spanish patients with presumed hereditary SNHL not caused by GJB2/GJB6, OTOF nor MT-RNR1 mutations.ResultsThe analytical sensitivity of the test to detect SNVs and indels on the DNA mixture from the cell lines was > 99.5%, with a specificity > 99.9%. The diagnostic yield on the SNHL patients was 42% (21/50): 47.6% (10/21) with autosomal recessive inheritance pattern (BSND, CDH23, MYO15A, STRC [n = 2], USH2A [n = 3], RDX, SLC26A4); 38.1% (8/21) autosomal dominant (ACTG1 [n = 3; 2 de novo], CHD7, GATA3 [de novo], MITF, P2RX2, SOX10), and 14.3% (3/21) X-linked (COL4A5 [de novo], POU3F4, PRPS1). 46.9% of causative variants (15/32) were not in the databases. 28.6% of genetically diagnosed cases (6/21) had previously undetected syndromes (Barakat, Usher type 2A [n = 3] and Waardenburg [n = 2]). 19% of genetic diagnoses (4/21) were attributable to large deletions/duplications (STRC deletion [n = 2]; partial CDH23 duplication; RDX exon 2 deletion).ConclusionsIn the era of precision medicine, obtaining an etiologic diagnosis of SNHL is imperative. Here, we contribute to show that, with the right methodology, NGS can be transferred to the clinical practice, boosting the yield of SNHL genetic diagnosis to 50–60% (including GJB2/GJB6 alterations), improving diagnostic/prognostic accuracy, refining genetic and reproductive counseling and revealing clinically relevant undiagnosed syndromes.Electronic supplementary materialThe online version of this article (10.1186/s12920-018-0375-5) contains supplementary material, which is available to authorized users.
The presence of Y chromosome sequences in Ullrich-Turner syndrome (UTS) patients has been suggested in previous work. Karyotype analysis estimated at about 60% of patients with a 45, X constitution and molecular analysis (Southern blot analysis with several Y chromosome probes and PCR of specific sequences) identified the presence of Y chromosome material in about 40% of 45, X patients. We have developed a very sensitive, PCR-based method to detect Y specific sequences in DNA from UTS patients. This protocol permits the detection of a single cell carrying a Y sequence among 10(5) Y-negative cells. We studied 18 UTS patients with 4 Y-specific sequences. In 11 patients we detected a positive amplification for at least one Y sequence. The existence of a simple and sensitive method for the detection of Y sequences has important implications for UTS patients, in view of the risk for some of the females carrying Y-chromosome material of developing gonadoblastoma and virilization. Additionally, some of the UTS associated phenotypes, such as renal anomalies, could be correlated with the presence of Y chromosome specific sequences.
PurposePatients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients’ characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies.Methods68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies.ResultsWe identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as ‘affecting functions’ are SNPs. Deep analysis of sequencing data revealed patients’ true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations)ConclusionWES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.
Purpose In the era of precision medicine, genomic characterization of blind patients is critical. Here, we evaluate the effects of comprehensive genetic analysis on the etiologic diagnosis of potentially hereditary vision loss and its impact on clinical management. Methods We studied 100 non‐syndromic and syndromic Spanish patients with a clinical diagnosis of blindness caused by alterations on the retina, choroid, vitreous and/or optic nerve. We used a next‐generation sequencing (NGS) panel (OFTALMOgenics™), developed and validated within this study, including up to 362 genes previously associated with these conditions. Results We identified the genetic cause of blindness in 45% of patients (45/100). A total of 28.9% of genetically diagnosed cases (13/45) were syndromic and, of those, in 30.8% (4/13) extraophthalmic features had been overlooked and/or not related to visual impairment before genetic testing, including cases with Mainzer‐Saldino, Bardet‐Biedl, mucolipidosis and MLCRD syndromes. In two additional cases–syndromic blindness had been proposed before, but not specifically diagnosed, and one patient with Heimler syndrome had been misdiagnosed as an Usher case before testing. 33.3% of the genetically diagnosed patients (15/45) had causative variants in genes targeted by clinical trials exploring the curative potential of gene therapy approaches. Conclusion Comprehensive genomic testing provided clinically relevant insights in a large proportion of blind patients, identifying potential therapeutic opportunities or previously undiagnosed syndromes in 42.2% of the genetically diagnosed cases (19/45).
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