CEP290 Research Articles

A novel mutation in CDK5RAP2 gene causes primary microcephaly with speech impairment and sparse eyebrows in a consanguineous Pakistani family

CDK5RAP2 gene encodes a centrosomal protein, highly expressed in fetal brain and essentially indispensable for its normal development, as biallelic mutations in it lead to primary microcephaly (MCPH). Despite being known as MCPH linked gene for more than a decade, the phenotypic spectrum of CDK5RAP2 mutations is still under explored as only eleven families have been reported worldwide. Here, we analyzed a consanguineous Pakistani MCPH family, characterized by moderate to severe intellectual disability, speech impairment, moderately short stature and sparse eyebrows. Whole exome sequencing of the proband identified a 2bp duplication in exon 34 of CDK5RAP2 that causes frame-shift, leading to a premature stop codon. The resultant transcript is resistant to nonsense mediated decay, suggesting that the mutation leads to a truncated protein lacking C-terminal domains; CDK5R1, and Cnn motif 2 (CM2), required for its localization to centrosome and Golgi Apparatus. Clinical variability observed in the family highlights the importance of further detailed clinical description of patients with CDK5RAP2 mutations.

Genetic research advance on neurodegeneration with brain iron accumulation

Neurodegeneration with brain iron accumulation (NBIA) is a neurodegenerative disorder characterized by abnormal accumulation of iron in central nervous system. Common clinical symptoms in NBIA include different types of dyskinesia, pyramidal tract involvement, cerebellar ataxia, peripheral neuropathy, autonomic neuropathy, cognitive impairment and visual dysfunction. So far, 10 genes have been identified as the causative gene for NBIA subtypes, which are PANK2, COASY, PLA2G6, C19orf12, FA2H, WDR45, ATP13A2, FTL, CP and DCAF17. The pathogenesis of NBIA involves mitochondrial involvement, oxidative stress damage, lipid metabolism and autophagy. Furthermore, NBIA may share the same pathogenetic mechanism with some other neurodegenerative disorders, such as Parkinson's disease (PD), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). DOI: 10.3969/j.issn.1672-6731.2017.07.004

An elusive ciliopathy: Joubert syndrome

The police brought a 65-year-old female patient to the EADU after being found ‘roaming the streets’ in an apparent state of confusion. This was her third admission under the same...

Japanese WDR45 de novo mutation diagnosed by exome analysis: A case report.

A 40‐year‐old Japanese woman presented with slowly progressing parkinsonism in adulthood. She had a history of epilepsy with intellectual disability in childhood. In a head magnetic resonance scan, T2‐weighted imaging showed low signal intensity areas in the globus pallidus and the substantia nigra; T1‐weighted imaging showed a halo in the nigra. Because the patient's symptoms and history were similar to those of patients with neurodegeneration with brain iron accumulation, we ran an exome analysis to investigate neurodegeneration with brain iron accumulation‐associated genes. We identified a c.700 C>T (p.Arg 234*) mutation in exon 9 of the WDR45 gene, which had not been reported in Japanese patients with beta‐propeller protein‐associated neurodegeneration (a neurodegeneration with brain iron accumulation subtype). Sanger sequencing confirmed a heterozygous mutation in this patient that was absent in both her parents, so it was judged to be a de novo nonsense mutation.

Evaluation of the association of single nucleotide polymorphisms in DDP4 and CDK5RAP2 genes with rheumatoid arthritis susceptibility in Iranian population

BackgroundRheumatoid arthritis (RA) is known as a chronic autoimmune inflammatory disorder, which is characterized mainly by the progressive inflammation and destruction of the joints. In the pathogenesis of RA, a variety of cell types such as lymphocyte, dendritic cells, osteoclasts and synovial fibroblasts are involved. Genetic proneness has been implicated in the pathogenesis of RA. The aim of this study was to evaluate the association of single nucleotide polymorphisms (SNPs) in DPP4 and CDK5RAP2 genes and risk of RA in Iranian population. MethodsFor genotyping, 623 RA patients and 412 healthy subjects were recruited. Genetic analysis of DPP4 gene rs12617656 and CDK5RAP2 gene rs12379034 polymorphisms was conducted using TaqMan allelic discrimination (for rs12617656) and ARMS-PCR (for rs12379034) methods. ResultsExperiments demonstrated that alleles and genotypes of both SNPs were represented equally in RA patients and controls. Statistical analysis revealed that none of the rs12617656 and rs12379034SNPs had significant differences in prevalence of both alleles and genotypes between RA patients and healthy controls. ConclusionsIt appears that gene polymorphisms of DPP4 and CDK5RAP2 are not involved in the pathogenesis of RA in Iranian population.

Whole-exome sequencing and digital PCR identified a novel compound heterozygous mutation in the NPHP1 gene in a case of Joubert syndrome and related disorders

BackgroundJoubert syndrome and related disorders (JSRD) is a clinically and genetically heterogeneous condition with autosomal recessive or X-linked inheritance, which share a distinctive neuroradiological hallmark, the so-called molar tooth sign. JSRD is classified into six clinical subtypes based on associated variable multiorgan involvement. To date, 21 causative genes have been identified in JSRD, which makes genetic diagnosis difficult.Case presentationWe report here a case of a 28-year-old Japanese woman diagnosed with JS with oculorenal defects with a novel compound heterozygous mutation (p.Ser219*/deletion) in the NPHP1 gene. Whole-exome sequencing (WES) of the patient identified the novel nonsense mutation in an apparently homozygous state. However, it was absent in her mother and heterozygous in her father. A read depth-based copy number variation (CNV) detection algorithm using WES data of the family predicted a large heterozygous deletion mutation in the patient and her mother, which was validated by digital polymerase chain reaction, indicating that the patient was compound heterozygous for the paternal nonsense mutation and the maternal deletion mutation spanning the site of the single nucleotide change.ConclusionIt should be noted that analytical pipelines that focus purely on sequence information cannot distinguish homozygosity from hemizygosity because of its inability to detect large deletions. The ability to detect CNVs in addition to single nucleotide variants and small insertion/deletions makes WES an attractive diagnostic tool for genetically heterogeneous disorders.

Juvenile nephronophthisis and dysthyroidism: a rare association.

Nephronophthisis, an autosomal recessive kidney disease, represents the most frequent genetic cause of end-stage kidney disease in the first three decades of life. A 27-year-old male was presented with gait imbalance, sever pruritus since 10days prior time of admission. In past medical history, he had bilateral cataract, torsional nystagmus, and bilateral optic nerve atrophy since 2years of age. He was also mentioned history of multinodular goiter with dysfunctional thyroid state since 2years before admission. At admission bilateral blindness, torsional nystagmus, asymmetric thyromegaly with nodularity was found in physical examination. Laboratory tests showed elevated urea and creatinine (200, 10.7mg/dl), hypomagnesemia (1.1mEq/l), decreased thyroid stimulating hormone (<0.004mIU/l). Ophthalmologist consultation confirmed retinitis pigmentosa. Renal sonography showed small-sized kidneys. Brain magnetic resonance imaging did not reveal molar tooth sign. Genetic testing performed and a large homozygous deletion at the NPHP1 gene locus was found. The patient was diagnosed with juvenile nephronophthisis and consideration of dysthyroidism as extrarenal manifestation of nephronophthisis is suggested in this case. Furthermore, loss of function mutation in SLC41A1 gene that leads to magnesium depletion must be noted in patients with suspected to nephronophthisis.

Ferrous Iron Up-regulation in Fibroblasts of Patients with Beta Propeller Protein-Associated Neurodegeneration (BPAN).

Mutations in WDR45 gene, coding for a beta-propeller protein, have been found in patients affected by Neurodegeneration with Brain Iron Accumulation, NBIA5 (also known as BPAN). BPAN is a movement disorder with Non Transferrin Bound Iron (NTBI) accumulation in the basal ganglia as common hallmark between NBIA classes (Hayflick et al., 2013). WDR45 has been predicted to have a role in autophagy, while the impairment of iron metabolism in the different NBIA subclasses has not currently been clarified. We found the up-regulation of the ferrous iron transporter (-)IRE/Divalent Metal Transporter1 and down-regulation of Transferrin receptor in the fibroblasts of two BPAN affected patients with splicing mutations 235+1G>A (BPAN1) and 517_519ΔVal 173 (BPAN2). The BPAN patients showed a concomitant increase of intracellular ferrous iron after starvation. An altered pattern of iron transporters with iron overload is highlighted in BPAN human fibroblasts, supporting for a role of DMT1 in NBIA. We here present a novel element, about iron accumulation, to the existing knowledge in field of NBIA. Attention is focused to a starvation-dependent iron overload, possibly accounting for iron accumulation in the basal ganglia. Further investigation could clarify iron regulation in BPAN.

Genome-Wide Detection of Copy Number Variations in Unsolved Inherited Retinal Disease.

Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of Mendelian disorders that plays a crucial role in the etiology of blindness across the world. Molecular genetic diagnosis of IRD remains extremely complex and challenging because mutations are only detected in 40% to 60% of cases. In this study, we aimed to dissect the contributions of copy number variations (CNVs) in IRD patients. A total of 50 patients were diagnosed with IRD, all of whom previously tested negative for pathogenic mutations in known disease genes. Single-nucleotide polymorphism array analysis was performed by using the HumanCoreExome BeadChip. Analyses of CNVs were carried out by using GenomeStudio, KaryoStudio, and cnvPartition. The putative pathogenic CNVs were further confirmed by real-time quantitative PCR. We identified four novel CNVs in three different genes (one duplication in USH2A gene, two duplications in CEP290 gene, and one duplication in RIMS2 gene) in total four families, at a detection rate of 8% (4/50). All of these CNVs are currently absent in all databases. Three variations are located in genes that are already known to cause inherited retinal disease: USH2A and CEP290, while the association between mutation in the RIMS2 gene and IRD is reported for the first time. We performed whole-genome-wide CNV analyses in a large cohort as an alternative approach to molecular diagnosis of IRDs. This study dissected the contributions of CNVs of IRDs, not only increasing the yield in genetic testing but also suggesting the CNVs should be analyzed in the patients with IRDs.

CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10

As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290 splice mutation. We generated a cellular model of LCA10 by introducing the CEP290 splice mutation into 293FT cells and we showed that guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that a dual AAV system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina. To minimize the immune response to prolonged expression of SpCas9, we developed a self-limiting CRISPR/Cas9 system that minimizes the duration of SpCas9 expression. These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of patients with LCA10.

Clinical features and gene mutation analysis of 13 Chinese juvenile patients with nephronophthisis

Objective: To explore the clinical features and pathogenic gene mutation of juvenile nephronophthisis (NPHP) in Chinese patients. Method: Clinical data and blood samples of 27 juvenile NPHP patients from 25 families who were initially clinically diagnosed in six hospitals in Guangdong province were collected. NPHP1 homozygous deletions were detected in all patients. Sequencing of NPHP1 gene was performed when homozygous deletions were not found in patients without eye involvement. In patients with eye involvement, NPHP5 sequencing was carried out initially and subsequently NPHP10 gene and NPHP1 when there were no NPHP5 gene mutation found. Result: Diagnosis was confirmed in 13 patients by renal pathology and (or) gene sequencing, including four boys and nine girls with a median onset age of 8.5(0.1-12.8) years. Seven of the 13 patients had a normal routine urine test and six patients had mild to moderate proteinuria. None had persistent hematuria. The estimated glomerular filtration rate of the 13 patients was (12.7±10.7) ml/(min·1.73 m2) at the time of diagnosis. Renal cysts were found in only five patients by iconography. Decreased renal size was observed in nine cases and normal renal size in four patients. Renal pathology was available in five patients, renal cysts formation at the cortical-medullar area, thickening and laying tubular basement membrane, were observed. Two of the thirteen children had eye involvement, one had liver impairment and one had growth retardation. NPHP1 gene defects were detected in seven patients with a mutation rate of 25.9%, and large homozygous deletions were observed in three patients. Four patients had single point mutations, i. e. compound heterozygous mutations (c.13 C>T and c. 1520+ 5 G>A) in one patient; homozygous mutation in three patients, two patients were siblings from the same pedigree harbored c. 1756 C>T and the other one harbored c. 1298delA. NPHP5 gene homozygous mutation was found in one pedigree. The fourteen children without renal pathology and whose genetic tests were negative shared similar clinical features with the thirteen patients whose diagnosis were confirmed by gene mutation and (or) renal pathology. Conclusion: The onset of juvenile NPHP is insidious. Urine and renal iconography changes are mild or negative. The ratio of NPHP1 mutant patient is similar with previous reports, but the proportion of NPHP1 gene homozygous deletions is much lower and all of the NPHP1 gene single point mutations detected in this research were novel, which indicates a genetic discrepancy existed between Chinese NPHP patients and the western ones.

Epileptic spasms: a previously unreported manifestation of WDR45 gene mutation.

Epileptic spasms: a previously unreported manifestation of WDR45 gene mutation.

The genetic epidemiology of the form of microcephaly ascribed to mutation at the WDR62 locus.

The disorder associated with mutation in the WDR62 gene MCPH2 is taken as the prototype of a condition which has a recessive mode of inheritance. The mutant homozygote has relatively lower fitness defined by the selection coefficient. Formulae which relate the incidence of the disorder to the mutation rate and the gene frequency in equilibrium when some degree of inbreeding occurs in the population are given.

Application of targeted exome capture in identifying genetic mutations in ciliopathies

Objective To develop an approach based on next-generation sequencing to determine the genetic defects in ciliopathies precisely and effectively. Methods Six suspected cases of ciliopathies were enrolled from July 2009 to December 2014 in the PLA General Hospital. Cord blood or tissue of the probands and peripheral blood samples of all the parents were collected for karyotyping analysis. Tissue or blood DNA from the six cases was scanned on 113 kinds of genes related to ciliopathies using targeted exome capture with high throughput sequencing. Mutations detected from the related genes were confirmed by direct Sanger sequencing reactions. Results Karyotype was normal in all the six cases. Pathogenic genes were found in five cases, including PKHD1 mutation in two cases (case one with c.6091delG and c.865delC, and case two with c.11042T>G and c.5137G>T); TCTN2 mutation in one case (c.343G>T and c.1540C>T) and CEP290 mutation in one case (c.7328_7332del and c.4897C>T). All were compound heterozygous mutations. TMEM67 mutation was found in one case (c.1645C>T), which was homozygous mutation. First-generation sequencing confirmed that these mutations originated from their parents. No definite pathogenic courses were found in one case (case six). Conclusions Targeted exome capture with high throughput sequencing is a new approach to find pathogenic gene mutations in ciliopathies. The methodology provides a reliable strategy for routine gene diagnosis and genetic counseling on ciliopathies. Key words: Polycystic kidney, autosomal recessive; Ciliary motility disorders; Encephalocele; Abnormalities, multiple; Exons; High-throughput nucleotide sequencing

Genome-Wide Analysis Identifies Germ-Line Risk Factors Associated with Canine Mammary Tumours.

Canine mammary tumours (CMT) are the most common neoplasia in unspayed female dogs. CMTs are suitable naturally occurring models for human breast cancer and share many characteristics, indicating that the genetic causes could also be shared. We have performed a genome-wide association study (GWAS) in English Springer Spaniel dogs and identified a genome-wide significant locus on chromosome 11 (praw = 5.6x10-7, pperm = 0.019). The most associated haplotype spans a 446 kb region overlapping the CDK5RAP2 gene. The CDK5RAP2 protein has a function in cell cycle regulation and could potentially have an impact on response to chemotherapy treatment. Two additional loci, both on chromosome 27, were nominally associated (praw = 1.97x10-5 and praw = 8.30x10-6). The three loci explain 28.1±10.0% of the phenotypic variation seen in the cohort, whereas the top ten associated regions account for 38.2±10.8% of the risk. Furthermore, the ten GWAS loci and regions with reduced genetic variability are significantly enriched for snoRNAs and tumour-associated antigen genes, suggesting a role for these genes in CMT development. We have identified several candidate genes associated with canine mammary tumours, including CDK5RAP2. Our findings enable further comparative studies to investigate the genes and pathways in human breast cancer patients.

124. Therapeutic Correction of an LCA-Causing Splice Defect in the CEP290 Gene by CRISPR/Cas-Mediated Gene Editing

124. Therapeutic Correction of an LCA-Causing Splice Defect in the CEP290 Gene by CRISPR/Cas-Mediated Gene Editing

329. Using CRISPR/Cas9 as a Therapeutic Approach for Leber Congenital Amaurosis 10 (LCA10)

Introduction: Leber congenital amaurosis (LCA) is the most severe form of inherited retinal diseases with early onset of symptoms in the first year of life. The most frequent genetic cause of LCA, accounting for approximately 15% of all LCA cases in western countries, is a deep-intronic mutation c. 2991+1655A>G located in the intron 26 of human CEP290 gene. LCA caused by CEP290 mutation is known as LCA10. The intronic mutation of CEP290 generates a cryptic splice donor site, resulting in the inclusion of a pseudoexon that leads to a premature stop codon and a truncated protein. The size of human CEP290 cDNA (~7.4 kb) exceeds the cargo size (~4.8 kb) of recombinant adeno-associated viral vectors (rAAVs), which makes this gene challenging for gene replacement therapy. In this regard, we tested if we can use the new genome editing technology CRISPR/Cas9 as an alternative strategy for LCA10 by removing the intronic mutation of CEP290 and preventing the cryptic splicing.Methods: CRISPR/SpCas9 was employed to introduce the intronic splice mutation c. 2991+1655 A>G into HEK 293FT cells to create a cellular model for LCA10/CEP290. In this cellular model, we used guide RNA pairs coupled with SpCas9 to delete the intronic region flanking the intronic mutation.Results: The deep-intronic mutation c. 2991+1655 A>G was successfully introduced into 293FT cells through the homology directed repair (HDR) pathway of CRISPR/spCas9. CEP290 expression levels were markedly reduced in the mutant cell line. Using this cellular model, we identified three sgRNA pairs that could efficiently (>50%) delete the intronic mutation, significantly rescue wild-type CEP290 expression levels and reduce mutant CEP290 expression.Conclusions: Our results demonstrate that the paired sgRNAs coupled with SpCas9 are capable of removing the c. 2991+1655A>G mutation and are highly efficient in preventing the splicing of the mutant cryptic exon and restoring wild-type CEP290 expression.

WDR45 mutations in Rett (-like) syndrome and developmental delay: Case report and an appraisal of the literature

Mutations in the WDR45 gene have been identified as causative for the only X-linked type of neurodegeneration with brain iron accumulation (NBIA), clinically characterized by global developmental delay in childhood, followed by a secondary neurological decline with parkinsonism and/or dementia in adolescence or early adulthood. Recent reports suggest that WDR45 mutations are associated with a broader phenotypic spectrum. We identified a novel splice site mutation (c.440-2 A > G) in a 5-year-old Argentinian patient with Rett-like syndrome, exhibiting developmental delay, microcephaly, seizures and stereotypic hand movements, and discuss this finding, together with a review of the literature. Additional patients with a clinical diagnosis of Rett (-like) syndrome were also found to carry WDR45 mutations before (or without) clinical decline or signs of iron accumulation by magnetic resonance imaging (MRI). This information indicates that WDR45 mutations should be added to the growing list of genetic alterations linked to Rett-like syndrome. Further, clinical symptoms associated with WDR45 mutations ranged from early-onset epileptic encephalopathy in a male patient with a deletion of WDR45 to only mild cognitive delay in a female patient, suggesting that analysis of this gene should be considered more often in patients with developmental delay, regardless of severity. The increasing use of next generation sequencing technologies as well as longitudinal follow-up of patients with an early diagnosis will help to gain additional insight into the phenotypic spectrum associated with WDR45 mutations.

Comparative Genomic Analyses of the Human NPHP1 Locus Reveal Complex Genomic Architecture and Its Regional Evolution in Primates.

Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100) is an autosomal recessive disorder that can be caused by defects of NPHP1; the gene maps within the human 2q13 region where low copy repeats (LCRs) are abundant. Loss of function of NPHP1 is responsible for approximately 85% of the NPHP1 cases—about 80% of such individuals carry a large recurrent homozygous NPHP1 deletion that occurs via nonallelic homologous recombination (NAHR) between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the NPHP1 gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV) haplotypes at the NPHP1 locus that may protect a haploid genome from the NPHP1 deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the NPHP1 locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the NPHP1 deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual’s susceptibility to acquiring disease-associated alleles.

Novel Alternative Splice Variants of Mouse Cdk5rap2.

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.