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.
"(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. "
[Show abstract][Hide abstract] ABSTRACT: Objective:
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.
In cooperation with academic societies, we analyzed the genomic copy number aberrations using chromosomal microarray testing. Finally, the genotype-phenotype correlation among them was examined.
We obtained clinical information of 86 patients who had been diagnosed with chromosomal deletions in the 1p36 region. Among them, blood samples were obtained from 50 patients (15 males and 35 females). The precise deletion regions were successfully genotyped. There were variable deletion patterns: pure terminal deletions in 38 patients (76%), including three cases of mosaicism; unbalanced translocations in seven (14%); and interstitial deletions in five (10%). Craniofacial/skeletal features, neurodevelopmental impairments, and cardiac anomalies were commonly observed in patients, with correlation to deletion sizes.
The genotype-phenotype correlation analysis narrowed the region responsible for distinctive craniofacial features and intellectual disability into 1.8-2.1 and 1.8-2.2 Mb region, respectively. Patients with deletions larger than 6.2 Mb showed no ambulation, indicating that severe neurodevelopmental prognosis may be modified by haploinsufficiencies of KCNAB2 and CHD5, located at 6.2 Mb away from the telomere. Although the genotype-phenotype correlation for the cardiac abnormalities is unclear, PRDM16, PRKCZ, and RERE may be related to this complication. Our study also revealed that female patients who acquired ambulatory ability were likely to be at risk for obesity.
Brain and Development 08/2014; 37(5). DOI:10.1016/j.braindev.2014.08.002 · 1.88 Impact Factor
"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. "
[Show abstract][Hide abstract] 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 · 5.14 Impact Factor
"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. "
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