Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy
ABSTRACT Nephronophthisis associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity, a strategy of DNA pooling with consecutive massively parallel resequencing (MPR) was devised.
In 120 patients with severe NPHP-AC phenotypes, five pools of genomic DNA with 24 patients each were prepared which were used as templates in order to PCR amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on an Illumina Genome-Analyser and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease based heteroduplex screening and confirmed by Sanger sequencing.
For proof of principle, DNA from patients with known mutations was used and detection of 22 out of 24 different alleles (92% sensitivity) was demonstrated. MPR led to the molecular diagnosis in 30/120 patients (25%) and 54 pathogenic mutations (27 novel) were identified in seven different NPHP-AC genes. Additionally, in 24 patients only single heterozygous variants of unknown significance were found.
The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single gene disorders. The lack of mutations in 75% of patients in this cohort indicates further extensive heterogeneity in NPHP-AC.
- SourceAvailable from: Kazuhiro Haginoya
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- "(NPHP4), is located on 1p36 (chr1: 5,946,555– 5,965,543) , proximal to the deletion region of three patients with renal abnormalities (Pt 26, 33, and 35). It is unclear whether there is a correlation between NPHP4 and the renal abnormalities observed in this study. "
ABSTRACT: Monosomy 1p36 syndrome is the most commonly observed subtelomeric deletion syndrome. Patients with this syndrome typically have common clinical features, such as intellectual disability, epilepsy, and characteristic craniofacial features.Brain and Development 08/2014; 37(5). DOI:10.1016/j.braindev.2014.08.002 · 1.54 Impact Factor
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- "Recently, others have also reported on the use of NGS for the identification of disease-causing mutations in genetically heterogeneous disorders, including that of dominant [Bowne et al., 2011] and recessive [Simpson et al., 2011] RP, although the number of genes that were analyzed and/or the cohort sizes were considerably smaller as compared with our study [Shearer et al., 2010; Jones et al., 2011; Otto et al., 2011]. An alternative for parallel, targeted resequencing of known disease genes is whole-exome or whole-genome sequencing [Choi et al., 2009; Lupski et al., 2010; Worthey et al., 2011], permitting a standardized laboratory workflow that can be used for any type of genetic disorder. "
ABSTRACT: Molecular diagnostics for patients with retinitis pigmentosa (RP) has been hampered by extreme genetic and clinical heterogeneity, with 52 causative genes known to date. Here, we developed a comprehensive next-generation sequencing (NGS) approach for the clinical molecular diagnostics of RP. All known inherited retinal disease genes (n = 111) were captured and simultaneously analyzed using NGS in 100 RP patients without a molecular diagnosis. A systematic data analysis pipeline was developed and validated to prioritize and predict the pathogenicity of all genetic variants identified in each patient, which enabled us to reduce the number of potential pathogenic variants from approximately 1,200 to zero to nine per patient. Subsequent segregation analysis and in silico predictions of pathogenicity resulted in a molecular diagnosis in 36 RP patients, comprising 27 recessive, six dominant, and three X-linked cases. Intriguingly, De novo mutations were present in at least three out of 28 isolated cases with causative mutations. This study demonstrates the enormous potential and clinical utility of NGS in molecular diagnosis of genetically heterogeneous diseases such as RP. De novo dominant mutations appear to play a significant role in patients with isolated RP, having major implications for genetic counselling.Human Mutation 08/2013; 33(6):963-72. DOI:10.1002/humu.22045 · 5.05 Impact Factor
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- "Many of the phenotypes tend to cluster around the type of cell affected in a particular organ. Renal failure that can begin in early childhood and progress into adolescence in NPHP is thought to result from kidney tubular basement membrane disintegration, tubular atrophy, and multiple cyst formations [Chaki et al., 2011; Otto et al., 2011]. Nephronophthis is due to homozygous or compound heterozygous mutations of INVS/NPHP2 typically diagnosed at a year of age, with the presence of hypertension and a concomitant or subsequent rather rapid onset of renal failure. "
ABSTRACT: Nephronophthisis associated ciliopathies (NPHP-AC) are a group of phenotypically related conditions that include Joubert syndrome, Meckel syndrome, nephronophthisis (NPHP), and Senior-Loken syndrome. We report on a male fetus with prenatal ultrasound findings at 24 weeks of gestation of anhydramnios, large and echogenic kidneys and situs inversus totalis. Histopathology revealed nephronophthisis and tracheal mucosa electron microscopy revealed ciliary dysgenesis. DNA analysis of the NPHP genes showed a previously unreported homozygous mutation, p.Arg603* (c.1078+1G>A), in the INVS/NPHP2 gene. This mutation is thought to abolish the splice donor site for exon 8, which likely disrupts the normal splicing of the INVS/NPHP2 gene. © 2013 Wiley Periodicals, Inc.American Journal of Medical Genetics Part A 07/2013; 161(7). DOI:10.1002/ajmg.a.36036 · 2.05 Impact Factor