Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing

Neurofibromatosis type 1 (NF1) is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.     

Frequence and the mutation spectrum of <Emphasis Type="Italic">GJB2</Emphasis>-related hearing loss in children of Dagestan as compared with the central European part of Russia

The sequencing of the entire coding region and the donor site of the splicing of the GJB2 gene has shown that prelingual neurosensory nonsyndromic autosomal recessive deafness/poor hearing in Dagestan Republic is genetically more heterogeneous than in the central European part of Russia. Thus, the number of the revealed mutations in the GJB2 gene in Dagestan was only 28% of the total number of alleles in patients tested. The main mutations in the GJB2 gene in Dagestan were represented by three forms typical for West Asia: 35delG mutation (22% of all mutant alleles), ΔE120 mutation (22%), both in the coding region, and IVS 1+1 G>A (44%) in the donor site of splicing. In addition, a novel mutation, ΔE187, was revealed in the coding region. All mutations were found in a compound heterozygous or a heterozygous state. This result allows one to explain the deafness/poor hearing by pathogenic mutations in the GJB2 gene only in 19% of Dagestan families with neurosensory nonsyndromic autosomal recessive disorders of hearing. We suggest that the origin and maintenance of the allele diversity of inherited deafness/poor hearing in Dagestan can be explained by the national and cultural peculiarities of small populations of nations of North Caucasus.     

No correlation between <Emphasis Type="Italic">NF1</Emphasis> mutation position and risk of optic pathway glioma in 77 unrelated NF1 patients

Neurofibromatosis type 1 (NF1) is a common monogenic disorder whereby affected individuals are predisposed to developing CNS tumors, including optic pathway gliomas (OPGs, occurring in ~15 to 20 % of cases). So far, no definite genotype–phenotype correlation determining NF1 patients at risk for tumor formation has been described, although enrichment for mutations in the 5′ region of the NF1 gene in OPG patients has been suggested. We used whole exome sequencing, targeted sequencing, and copy number analysis to screen 77 unrelated NF1 patients with (n = 41) or without (n = 36; age ≥10 years) optic pathway glioma for germline NF1 alterations. We identified germline NF1 mutations in 69 of 77 patients (90 %), but no genotype–phenotype correlation was observed. Our data using a larger patient cohort did not confirm the previously reported clustering of mutations in the 5′ region of the NF1 gene in patients with OPG. Thus, NF1 mutation location should not currently be used as a clinical criterion to assess the risk of developing OPGs.     

Two Novel Mutations in LAMC2 Gene in Iranian Families Affected by Junctional Epidermolysis Bullosa

Background:Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin disorder with defective adhesion of dermal- epidermal within the lamina lucida region of the basement membrane zone. The main characterization of JEB is blistering and fragile skin and mucous membrane. Laminins are noncollagenous part of basement membrane and classified as a family of extracellular matrix glycoprotein. Laminins contain three chains: Laminin α, Laminin β and Laminin γ. LAMC2 (laminin subunit gamma 2) gene encodes γ subunit of laminin and its mutation contributes to JEB. Here, we report a disease-causing nonsense mutation and a large deletion mutation in LAMC2 gene in two families affected by JEB.Methods:Whole exome sequencing (WES) was carried out on the mother of patient in family I and the patient himself in family II to detect the underlying mutations. Then, sanger sequencing was performed to confirm the identified mutations.Results:Next generation sequencing (NGS) data analysis of the first family showed a novel, nonsense mutation in LAMC2 gene (LAMC2: {"type":"entrez-nucleotide","attrs":{"text":"NM_005562","term_id":"1653960840"}}NM_005562: exon14:c.C2143T: p.R715X). The heterozygous state of the mutation was confirmed by sanger sequencing in the parents and unaffected brother. In Family II, NGS data had no coverage in the large area of LAMC2 gene. Thus, to confirm the possible deletion sanger sequencing was done and blasting of sequence showed the deleted region of 9.4 kb (exon10-17) in LAMC2 gene.Conclusion:In summary, current study reported a novel disease-causing premature termination codon (PTC) mutation in LAMC2 gene and a large deletion mutation in patients affected by JEB.Key Words: Junctional Epidermolysis Bullosa, LAMC2 gene, Novel mutation, Skin disorder     

Genetic and epigenetic alterations of the NF2 gene in sporadic vestibular schwannomas

Background

Mutations in the neurofibromatosis type 2 (NF2) tumor-suppressor gene have been identified in not only NF2-related tumors but also sporadic vestibular schwannomas (VS). This study investigated the genetic and epigenetic alterations in tumors and blood from 30 Korean patients with sporadic VS and correlated these alterations with tumor behavior.

Methodology/Principal Findings

NF2 gene mutations were detected using PCR and direct DNA sequencing and three highly polymorphic microsatellite DNA markers were used to assess the loss of heterozygosity (LOH) from chromosome 22. Aberrant hypermethylation of the CpG island of the NF2 gene was also analyzed. The tumor size, the clinical growth index, and the proliferative activity assessed using the Ki-67 labeling index were evaluated. We found 18 mutations in 16 cases of 30 schwannomas (53%). The mutations included eight frameshift mutations, seven nonsense mutations, one in-frame deletion, one splicing donor site, and one missense mutation. Nine patients (30%) showed allelic loss. No patient had aberrant hypermethylation of the NF2 gene and correlation between NF2 genetic alterations and tumor behavior was not observed in this study.

Conclusions/Significance

The molecular genetic changes in sporadic VS identified here included mutations and allelic loss, but no aberrant hypermethylation of the NF2 gene was detected. In addition, no clear genotype/phenotype correlation was identified. Therefore, it is likely that other factors contribute to tumor formation and growth.     

Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases

Background

Inherited retinal disorders are clinically and genetically heterogeneous with more than 150 gene defects accounting for the diversity of disease phenotypes. So far, mutation detection was mainly performed by APEX technology and direct Sanger sequencing of known genes. However, these methods are time consuming, expensive and unable to provide a result if the patient carries a new gene mutation. In addition, multiplicity of phenotypes associated with the same gene defect may be overlooked.

Methods

To overcome these challenges, we designed an exon sequencing array to target 254 known and candidate genes using Agilent capture. Subsequently, 20 DNA samples from 17 different families, including four patients with known mutations were sequenced using Illumina Genome Analyzer IIx next-generation-sequencing (NGS) platform. Different filtering approaches were applied to identify the genetic defect. The most likely disease causing variants were analyzed by Sanger sequencing. Co-segregation and sequencing analysis of control samples validated the pathogenicity of the observed variants.

Results

The phenotype of the patients included retinitis pigmentosa, congenital stationary night blindness, Best disease, early-onset cone dystrophy and Stargardt disease. In three of four control samples with known genotypes NGS detected the expected mutations. Three known and five novel mutations were identified in NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 and CACNA1F. One of the control samples with a known genotype belongs to a family with two clinical phenotypes (Best and CSNB), where a novel mutation was identified for CSNB. In six families the disease associated mutations were not found, indicating that novel gene defects remain to be identified.

Conclusions

In summary, this unbiased and time-efficient NGS approach allowed mutation detection in 75% of control cases and in 57% of test cases. Furthermore, it has the possibility of associating known gene defects with novel phenotypes and mode of inheritance.     

Targeted Exome Sequencing Integrated with Clinicopathological Information Reveals Novel and Rare Mutations in Atypical,Suspected and Unknown Cases of Alport Syndrome or Proteinuria

We applied customized targeted next-generation exome sequencing (NGS) to determine if mutations in genes associated with renal malformations, Alport syndrome (AS) or nephrotic syndrome are a potential cause of renal abnormalities in patients with equivocal or atypical presentation. We first sequenced 4,041 exons representing 292 kidney disease genes in a Caucasian woman with a history of congenital vesicoureteral reflux (VUR), recurrent urinary tract infections and hydronephrosis who presented with nephrotic range proteinuria at the age of 45. Her biopsy was remarkable for focal segmental glomerulosclerosis (FSGS), a potential complication of longstanding VUR. She had no family history of renal disease. Her proteinuria improved initially, however, several years later she presented with worsening proteinuria and microhematuria. NGS analysis revealed two deleterious COL4A3 mutations, one novel and the other previously reported in AS, and a novel deleterious SALL2 mutation, a gene linked to renal malformations. Pedigree analysis confirmed that COL4A3 mutations were nonallelic and compound heterozygous. The genomic results in conjunction with subsequent abnormal electron microscopy, Collagen IV minor chain immunohistochemistry and progressive sensorineural hearing loss confirmed AS. We then modified our NGS approach to enable more efficient discovery of variants associated with AS or a subset of FSGS by multiplexing targeted exome sequencing of 19 genes associated with AS or FSGS in 14 patients. Using this approach, we found novel or known COL4A3 or COL4A5 mutations in a subset of patients with clinically diagnosed or suspected AS, APOL1 variants associated with FSGS in African Americans and novel mutations in genes associated with nephrotic syndrome. These studies demonstrate the successful application of targeted capture-based exome sequencing to simultaneously evaluate genetic variations in many genes in patients with complex renal phenotypes and provide insights into etiology of conditions with equivocal clinical and pathologic presentations.     

Illegitimate splicing of the NF1 gene in healthy individuals mimics mutation-induced splicing alterations in NF1 patients

Neurofibromatosis type 1 (NF1) is a common inherited disease affecting one in 3,500 individuals. The mutation rate in the NF1 gene is one of the highest known for human genes. Compared to other methods, the protein truncation test (PTT) and subsequent sequence analysis of cloned cDNA provides improved efficiency in detecting NF1 mutations that are dispersed throughout the gene spanning 350 kb of genomic DNA. Sequencing of cDNA of patients affected with NF1 mutations revealed multiple splicing errors. Since similar missplicings were also found in "aged" blood of healthy individuals, they are most likely attributable to a general decrease in splice site selection in aged blood. We show that restoring viability of lymphocytes before RNA extraction by cultivation and PHA stimulation diminishes aberrant splicing in aged blood and is thus useful to circumvent splicing alterations which are frequently compromising mutation detection in patient samples and mimic mutation-induced alterations of mRNA.     

NPC1 gene mutations in Japanese patients with Niemann-Pick disease type C

Complementary and genomic DNAs isolated from the fibroblasts of 10 Japanese (7 late infantile, 2 juvenile, and 1 adult form of the disease) and one Caucasian patient with Niemann-Pick disease type C were analyzed for mutations in the NPC1 gene. Fourteen novel mutations were found including small deletions and point mutations. A one-base deletion and a point mutation caused splicing errors. The mutations were not clustered in any particular region of the gene and were found both in and out of the transmembrane domains. Three patients were homozygous, five were compound heterozygous, and the remaining three were suspected of being compound hetrozygous with an unknown error in one of their NPC1 alleles. Of the 14 mutations, the G1553A substitution that caused a splicing error of exon 9 appeared to be relatively common in Japanese patients, because two patients were homozygous and one patient was compound heterozygous for this mutation. Electronic Publication     

Identification of fibrillin-1 gene mutations in Marfan syndrome by high-resolution melting analysis

Marfan syndrome has been associated with approximately 562 mutations in the fibrillin-1 (FBN1) gene. Mutation scanning of the FBN1 gene with DNA direct sequencing is time-consuming and expensive because of its large size. This study analyzed the diagnostic value of high-resolution melting analysis as an alternative method for scanning of the FBN1 gene. A total of 75 polymerase chain reaction (PCR) amplicons (179-301 bp, average 256 bp) that covered the complete coding regions and splicing sites were evaluated on the 96-well LightCycler system. Melting curves were analyzed as fluorescence derivative plots (−dF/dT vs. temperature). To determine the sensitivity of this method, a total of 82 samples from patients with Marfan syndrome and 50 unaffected individuals were analyzed. All mutations reported in this study had been confirmed previously by direct sequencing analysis. Melting analysis identified 48 heterozygous variants. The variant c.3093 G>T (exon 25) was incorrectly identified by melting curve analysis. The sensitivity of the technique in this sample was 98.78% (81/82). This study demonstrated that high-resolution melting analysis is a reliable gene scanning method with greater speed than DNA sequencing. Our results support the use of this technology as an alternative method for the diagnosis of Marfan syndrome as well as its suitability for high-throughput mutation scanning of other large genes.     

Hermansky-Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage-pool deficiency

Hermansky-Pudlak syndrome (HPS), consisting of oculocutaneous albinism and a bleeding diathesis due to the absence of platelet dense granules, displays extensive locus heterogeneity. HPS1 mutations cause HPS-1 disease, and ADTB3A mutations cause HPS-2 disease, which is known to involve abnormal intracellular vesicle formation. A third HPS-causing gene, HPS3, was recently identified on the basis of homozygosity mapping of a genetic isolate of HPS in central Puerto Rico. We now describe the clinical and molecular characteristics of eight patients with HPS-3 who are of non-Puerto Rican heritage. Five are Ashkenazi Jews; three of these are homozygous for a 1303+1G-->A splice-site mutation that causes skipping of exon 5, deleting an RsaI restriction site and decreasing the amounts of mRNA found on northern blotting. The other two are heterozygous for the 1303+1G-->A mutation and for either an 1831+2T-->G or a 2621-2A-->G splicing mutation. Of 235 anonymous Ashkenazi Jewish DNA samples, one was heterozygous for the 1303+1G-->A mutation. One seven-year-old boy of German/Swiss extraction was compound heterozygous for a 2729+1G-->C mutation, causing skipping of exon 14, and resulting in a C1329T missense (R396W), with decreased mRNA production. A 15-year-old Irish/English boy was heterozygous for an 89-bp insertion between exons 16 and 17 resulting from abnormal splicing; his fibroblast HPS3 mRNA is normal in amount but is increased in size. A 12-year-old girl of Puerto Rican and Italian background has the 3,904-bp founder deletion from central Puerto Rico on one allele. All eight patients have mild symptoms of HPS; two Jewish patients had received the diagnosis of ocular, rather than oculocutaneous, albinism. These findings expand the molecular diagnosis of HPS, provide a screening method for a mutation common among Jews, and suggest that other patients with mild hypopigmentation and decreased vision should be examined for HPS.     

Identification of Novel GRM1 Mutations and Single Nucleotide Polymorphisms in Prostate Cancer Cell Lines and Tissues

Metabotropic glutamate receptor 1 (GRM1) signaling has been implicated in benign and malignant disorders including prostate cancer (PCa). To further explore the role of genetic alterations of GRM1 in PCa, we screened the entire human GRM1 gene including coding sequence, exon-intron junctions, and flanking untranslated regions (UTRs) for the presence of mutations and single nucleotide polymorphisms (SNPs) in several PCa cell lines and matched tumor-normal tissues from Caucasian Americans (CAs) and African Americans (AAs). We used bidirectional sequencing, allele-specific PCR, and bioinformatics to identify the genetic changes in GRM1 and to predict their functional role. A novel missense mutation identified at C1744T (582 Pro > Ser) position of GRM1 gene in a primary AA-PCa cell line (E006AA) was predicted to affect the protein stability and functions. Another novel mutation identified at exon-intron junction of exon-8 in C4-2B cell line resulted in alteration of the GRM1 splicing donor site. In addition, we found missense SNP at T2977C (993 Ser > Pro) position and multiple non-coding mutations and SNPs in 3′-UTR of GRM1 gene in PCa cell lines and tissues. These novel mutations may contribute to the disease by alterations in GRM1 gene splicing, receptor activation, and post-receptor downstream signaling.     

Mutations in RECQL Gene Are Associated with Predisposition to Breast Cancer

The genetic cause for approximately 80% of familial breast cancer patients is unknown. Here, by sequencing the entire exomes of nine early-onset familial breast cancer patients without BRCA1/2 mutations (diagnosed with breast cancer at or before the age of 35) we found that two index cases carried a potentially deleterious mutation in the RECQL gene (RecQ helicase-like; chr12p12). Recent studies suggested that RECQL is involved in DNA double-strand break repair and it plays an important role in the maintenance of genomic stability. Therefore, we further screened the RECQL gene in an additional 439 unrelated familial breast cancer patients. In total, we found three nonsense mutations leading to a truncated protein of RECQL (p.L128X, p.W172X, and p.Q266X), one mutation affecting mRNA splicing (c.395-2A>G), and five missense mutations disrupting the helicase activity of RECQL (p.A195S, p.R215Q, p.R455C, p.M458K, and p.T562I), as evaluated through an in vitro helicase assay. Taken together, 9 out of 448 BRCA-negative familial breast cancer patients carried a pathogenic mutation of the RECQL gene compared with one of the 1,588 controls (P = 9.14×10^-6). Our findings suggest that RECQL is a potential breast cancer susceptibility gene and that mutations in this gene contribute to familial breast cancer development.