USH2A Mutations Research Articles

Novel mutations in the USH2A gene in a family affected with Usher syndrome type 2

Objective To identify the pathogenic genes and mutations in a family with Usher syndrome type 2. Methods A three-generation family including 7 individuals was enrolled in this study. There were 2 male patients and 5 unaffected individuals. All participants was underwent related ophthalmologic examination, including best corrected visual acuity, slit-lamp, indirect ophthalmoscopy, electroretinogram (ERG), optical coherence tomography and visual field test. DNA was extracted from 3 ml peripheral venous blood of all participants. A total of 136 hereditary retinal disease target genes were screened and the DNA sequence was performed by Next-generation sequence analysis. Then the suspected mutations compared with databases to identify the suspected mutations, which should be verified with non-affected family members and 100 normal subjects by PCR and Sanger sequence. Results The sequence result showed that 2 patients, the proband and his brother, carried complex heterozygous mutations in the USH2A gene: c.5459T> C (p.M1820T) in exon 27, c.802G> A (p.G268R) in exon 5 and c.1 190T> A (p.I397K) in exon 7. The c.5459T> C and c.1190T> A mutations in USH2A have not been reported in the literature and database. Although their mother carried c.5459T> C (p.M1820T) and c.802G> A (p.G268R), and their father carried c.1190T> A (p.I397K) heterozygous mutations, the parents did not present phenotype. These mutations were not detected in other normal family members. The result was supported by co-segregation analysis. Conclusion The heterozygous mutations c.5459T> C (p.M1820T), c.1190T> A (p.I397K) and c.802G> A (p.G268R) in USH2A gene cause Usher syndrome in this family. Key words: Usher syndromes/genetics; Usher syndromes/etiology; Genes; Mutation; Sequence analysis

Usherin defects lead to early-onset retinal dysfunction in zebrafish

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

Comprehensive Molecular Screening in Chinese Usher Syndrome Patients.

Usher syndrome (USH) refers to a group of autosomal recessive disorders causing deafness and blindness. The objectives of this study were to determine the mutation spectrum in a cohort of Chinese patients with USH and to describe the clinical features of the patients with mutations. A total of 119 probands who were clinically diagnosed with USH were recruited for genetic analysis. All probands underwent ophthalmic examinations. A combination of molecular screening methods, including targeted next-generation sequencing, Sanger-DNA sequencing, and multiplex ligation probe amplification assay, was used to detect mutations. We found biallelic mutations in 92 probands (77.3%), monoallelic mutations in 5 patients (4.2%), and 1 hemizygous mutation in 1 patient (0.8%), resulting in an overall mutation detection rate of 78.2%. Overall, 132 distinct disease-causing mutations involving seven USH (ABHD12, CDH23, GPR98, MYO7A, PCDH15, USH1C, and USH2A) genes; 5 other retinal degeneration genes (CHM, CNGA1, EYS, PDE6B, and TULP1); and 1 nonsyndromic hearing loss gene (MYO15A) were identified, and 78 were novel. Mutations of MYOA7 were responsible for 60% of USH1 families, followed by PCDH15 (20%) and USH1C (10%). Mutations of USH2A accounted for 67.7% of USH2 families, and mutation c.8559-2A>G was the most frequent one, accounting for 19.1% of the identified USH2A alleles. Our results confirm that the mutation spectrum for each USH gene in Chinese patients differs from those of other populations. The formation of the mutation profile for the Chinese population will enable a precise genetic diagnosis for USH patients in the future.

The Genetics of Usher Syndrome in the Israeli and Palestinian Populations.

Usher syndrome (USH) is the most common cause for deaf-blindness. It is genetically and clinically heterogeneous and prevalent in populations with high consanguinity rate. We aim to characterize the set of genes and mutations that cause USH in the Israeli and Palestinian populations. Seventy-four families with USH were recruited (23 with USH type 1 [USH1], 33 with USH2, seven with USH3, four with atypical USH, and seven families with an undetermined USH type). All affected subjects underwent a full ocular evaluation. A comprehensive genetic analysis, including Sanger sequencing for the detection of founder mutations, homozygosity mapping, and whole exome sequencing in large families was performed. In 79% of the families (59 out of 74), an autosomal recessive inheritance pattern could be determined. Mutation detection analysis led to the identification of biallelic causative mutations in 51 (69%) of the families, including 21 families with mutations in USH2A, 17 in MYO7A, and seven in CLRN1. Our analysis revealed 28 mutations, 11 of which are novel (including c.802G>A, c.8558+1G>T, c.10211del, and c.14023A>T in USH2A; c.285+2T>G, c.2187+1G>T, c.3892G>A, c.5069_5070insC, c.5101C>T, and c.6196C>T in MYO7A; and c.15494del in GPR98). We report here novel homozygous mutations in various genes causing USH, extending the spectrum of causative mutations. We also prove combined sequencing techniques as useful tools to identify novel disease-causing mutations. To the best of our knowledge, this is the largest report of a genetic analysis of Israeli and Palestinian families (n = 74) with different USH subtypes.

Ultra-Widefield Fundus Autofluorescence Imaging of Patients with Retinitis Pigmentosa: A Standardized Grading System in Different Genotypes

To report a genotype-phenotype correlation study of patients with retinitis pigmentosa (RP) based on ultra-widefield (UWF) fundus autofluorescence (FAF) imaging. Case series. Thirty-four patients with RP. This retrospective study included RP patients with confirmed causative genetic variants and UWF FAF imaging data. Qualitative grading criteria including the pattern of macular abnormal autofluorescence, decreased autofluorescence (DAF), and its extent and distribution were applied to evaluate the genotype-phenotype correlation. The main parameters measured were increased or decreased patterns and extent of autofluorescence. Thirty-four unrelated patients 38±19 years of age (range, 9-82 years) were enrolled. Mutations in 17 different genes were detected in patients, including 7 patients having mutations in USH2A, 4 in DHDDS, 4 in RPGR, 3 in PRPF31, and 3 in RP1. Patients with nummular DAF and widespread DAF were significantly older (59±14 years and 56±19 years, respectively). All 3 patients with PRPF31 mutations showed an abnormal macular ring hyperautofluorescence and a circular pattern of coarse DAF distributed in Early Treatment Diabetic Retinopathy Study fields 1, 2, and 3 with sparing of the far periphery. In other genotypes, no specific DAF or macular abnormal autofluorescence pattern could be discerned. Specific UWF FAF characteristics in RP patients were correlated strongly with patient age and stage of the disease. Particular UWF FAF characteristics were found to be more prominent in a unique genotype.

STRC Deletion is a Frequent Cause of Slight to Moderate Congenital Hearing Impairment in the Czech Republic

This study aimed to clarify the molecular epidemiology of hearing loss by identifying the responsible genes in patients without GJB2 mutations. Prospective genetic study. Tertiary referral hospital. Fifty one patients with bilateral sensorineural hearing loss, 20 men, and 31 women, mean age 24.9 years, range 3 to 64 years, from 49 families. GJB2 and deltaGJB6-D13S1830 mutations were excluded previously. Diagnostic. Sixty-nine genes reported to be causative of hearing loss were analyzed. Sequence capture technology, next-generation sequencing, and multiplex ligation-dependent probe amplification (MLPA) were used. Coverage of STRC was screened in Integrative Genomics Viewer software. Identification of causal pathogenic mutations in genes related to deafness. Five families (10%) had recessive STRC deletions or mutations. Five unrelated patients (10%) had recessive mutations in TMPRSS3, USH2A, PCDH15, LOXHD1, and MYO15A. Three families (6%) had autosomal dominant mutations in MYO6A, KCNQ4, and SIX1. One family (2%) had an X-linked POU3F4 mutation. Thus, we identified the cause of hearing loss in 28% of the families studied. Following GJB2, STRC was the second most frequently mutated gene in patients from the Czech Republic with hearing loss. To decrease the cost of testing, we recommend STRC deletion screening with MLPA before next-generation sequencing. The existence of a pseudogene and polymorphic STRC regions can lead to false-positive or false-negative results when copy number variation analysis is based on next-generation sequencing data.

Targeted next generation sequencing in Italian patients with Usher syndrome: phenotype-genotype correlations

We report results of DNA analysis with next generation sequencing (NGS) of 21 consecutive Italian patients from 17 unrelated families with clinical diagnosis of Usher syndrome (4 USH1 and 17 USH2) searching for mutations in 11 genes: MYO7A, CDH23, PCDH15, USH1C, USH1G, USH2A, ADGVR1, DFNB31, CLRN1, PDZD7, HARS. Likely causative mutations were found in all patients: 25 pathogenic variants, 18 previously reported and 7 novel, were identified in three genes (USH2A, MYO7A, ADGRV1). All USH1 presented biallelic MYO7A mutations, one USH2 exhibited ADGRV1 mutations, whereas 16 USH2 displayed USH2A mutations. USH1 patients experienced hearing problems very early in life, followed by visual impairment at 1, 4 and 6 years. Visual symptoms were noticed at age 20 in a patient with homozygous novel MYO7A missense mutation c.849G > A. USH2 patients’ auditory symptoms, instead, arose between 11 months and 14 years, while visual impairment occurred later on. A homozygous c.5933_5940del;5950_5960dup in USH2A was detected in one patient with early deafness. One patient with homozygous deletion from exon 23 to 32 in USH2A suffered early visual symptoms. Therefore, the type of mutation in USH2A and MYO7A genes seems to affect the age at which both auditory and visual impairment occur in patients with USH.

The Genetic Basis of Pericentral Retinitis Pigmentosa-A Form of Mild Retinitis Pigmentosa.

Pericentral retinitis pigmentosa (RP) is an atypical form of RP that affects the near-peripheral retina first and tends to spare the far periphery. This study was performed to further define the genetic basis of this phenotype. We identified a cohort of 43 probands with pericentral RP based on a comprehensive analysis of their retinal phenotype. Genetic analyses of DNA samples from these patients were performed using panel-based next-generation sequencing, copy number variations, and whole exome sequencing (WES). Mutations provisionally responsible for disease were found in 19 of the 43 families (44%) analyzed. These include mutations in RHO (five patients), USH2A (four patients), and PDE6B (two patients). Of 28 putatively pathogenic alleles, 15 (54%) have been previously identified in patients with more common forms of typical RP, while the remaining 13 mutations (46%) were novel. Burden testing of WES data successfully identified HGSNAT as a cause of pericentral RP in at least two patients, suggesting it is also a relatively common cause of pericentral RP. While additional sequencing might uncover new genes specifically associated with pericentral RP, the current results suggest that genetically pericentral RP is not a separate clinical entity, but rather is part of the spectrum of mild RP phenotypes.

Visual Outcomes in Japanese Patients with Retinitis Pigmentosa and Usher Syndrome Caused by USH2A Mutations

ABSTRACTPurpose: EYS and USH2A are the most common causative genes for retinitis pigmentosa (RP) in Japan. We determined the clinical outcomes for USH2A-related non-syndromic RP or Usher syndrome type II (USH2). Methods: Two non-syndromic RP and 11 USH2 patients with previously identified USH2A mutations were included. Their complete history and medical records were collected using standard procedures. Visual fields and acuity were compared with those of patients with EYS mutations. Clinical analyses were based on ophthalmic and otolaryngologic examinations. Results: In all patients, the fundus displayed changes typical of RP. Most patients showed relatively well-preserved visual acuity in their thirties or forties, with rapid deterioration in their fifties. Concentric constriction started in the twenties or thirties, and no effective residual visual field was observed after the fifties. Conclusions: The visual outcome for non-syndromic RP or USH2 patients with USH2A mutations is consistent with that for RP patients with EYS mutations.

Molecular Analysis of Twelve Pakistani Families with Nonsyndromic or Syndromic Hearing Loss.

To investigate the causative genetic mutations in 12 Pakistani families with nonsyndromic or syndromic hearing loss. Mutations in the most common causative gene for hearing loss, GJB2, were evaluated by Sanger sequencing. Targeted next-generation sequencing or whole-exome sequencing was used to analyze the genomic DNA samples from 11 probands with hearing loss. Sanger sequencing was performed to verify all identified variants. We found pathogenic, or likely to be pathogenic, mutations in all 12 families, including six known mutations in GJB2, SLC26A4, LHFPL5, and USH2A and eight novel mutations in ESPN, MYO7A, LRTOMT, PCDH15, USH2A, or EPS8L2. Notably, four compound heterozygous mutations in the MYO7A and USH2A genes were detected in two consanguineous families. In addition, the novel frameshift mutation in EPS8L2 was first documented in Pakistan. Our study increases the spectrum of mutations associated with hearing loss in the Pakistani population. In addition, our study highlights the fact that compound heterozygous mutations, although rare, can occur in consanguineous families.

Analysis of mutations of disease-causing gene and the inheritance in sporadic retinitis pigmentosa

Objective To observe the disease-causing genes and the inheritance in sporadic retinitis pigmentosa (sRP) in Ningxia region. Methods 49 sRP patients and 128 family members were recruited for this study. All the patients and family members received complete ophthalmic examinations including best corrected visual acuity, slit-lamp microscope, indirect ophthalmoscopy, fundus color photography, visual field, optic coherence tomography, full view electroretinogram. DNA was extracted from patients and family members. Using exon combined target region capture sequencing chip to screen the 230 candidate disease-causing gene mutations, polymerase chain reaction and direct sequencing were used to confirm the disease-causing mutations. Results 24/49 patients (49.0%) had identified disease-causing genes, totally 16 genes were involved. There were 41 mutation sites were found, including 32 new mutations (78.0%). The disease-causing genes include USH2A, C2orf71, GNGA1, RPGR1, IFT140, TULP1, CLRN1, RPE65, ABCA4, GUCA1, EYS, CYP4V2, GPR98 and ATXN7. Based on pedigree analysis, 20 patients were autosomal recessive retinitis pigmentosa, 3 patients were autosomal dominant retinitis pigmentosa and 1 patient was X linked retinitis pigmentosa. 3/7 patients with USH2A mutations were identified as Usher syndrome. Conclusions USHZA is the main disease-causing of sRP patients in Ningxia region. 83.3% of sRP in this cohort are autosomal recessive retinitis pigmentosa. Key words: Retinitis pigmentosa/genetics; Inheritance patterns; Genes; Mutation; High-throughput nucleotide sequencing

Molecular genetic basis of Usher syndrome in the Czech population

SummaryA systematic literature search of the PubMed database (1 March 2016) using keywords ‘Usher syndrome’ AND ‘mutation’ AND ‘Czech Republic’ (or Belarus, Bulgaria, Estonia, Hungary, Latvia, Lithuania, Moldova, Poland, Slovakia, Romania, Russia, Ukraine) yielded 3 results. After inspection of retrieved abstracts we concluded that none of the published patients had an established molecular genetic cause of Usher syndrome. This is in striking contrast to numerous reports from Western European populations. In this presentation we will discuss our recently established program at a tertiary referral centre in Prague to genotype patients with Usher syndrome. So far 17 probands and 27 unaffected first degree relatives have been examined and donated DNA samples. The search into the molecular genetic causes is ongoing, however it is already apparent that there is an overrepresentation of the c.11864G>A; p.(Trp3955*) mutation in USH2A which was found on at least one allele in 9 probands (53%). The impact of our findings on clinical care and their potential to provide translational outcomes will be discussed. The spectrum of mutations identified in Czech Usher syndrome patients may also be relevant for neighbouring countries.

A combination of two truncating mutations in USH2A causes more severe and progressive hearing impairment in Usher syndrome type IIa

ObjectivesUsher syndrome is an inherited disorder that is characterized by hearing impairment (HI), retinitis pigmentosa, and in some cases vestibular dysfunction. Usher syndrome type IIa is caused by mutations in USH2A. HI in these patients is highly heterogeneous and the present study evaluates the effects of different types of USH2A mutations on the audiometric phenotype. Data from two large centres of expertise on Usher Syndrome in the Netherlands and Sweden were combined in order to create a large combined sample of patients to identify possible genotype-phenotype correlations. DesignA retrospective study on HI in 110 patients (65 Dutch and 45 Swedish) genetically diagnosed with Usher syndrome type IIa. We used methods especially designed for characterizing and testing differences in audiological phenotype between patient subgroups. These methods included Age Related Typical Audiograms (ARTA) and a method to evaluate the difference in the degree of HI developed throughout life between subgroups. ResultsCross-sectional linear regression analysis of last-visit audiograms for the best hearing ear demonstrated a gradual decline of hearing over decades. The congenital level of HI was in the range of 16–33 dB at 0.25–0.5 kHz, and in the range of 51–60 dB at 1–8 kHz. The annual threshold deterioration was in the range of 0.4–0.5 dB/year at 0.25–2 kHz and in the range of 0.7–0.8 dB/year at 4–8 kHz. Patients with two truncating mutations, including homozygotes for the common c.2299delG mutation, developed significantly more severe HI throughout life than patients with one truncating mutation combined with one nontruncating mutation, and patients with two nontruncating mutations. ConclusionsThe results have direct implications for patient counselling in terms of prognosis of hearing and may serve as baseline measures for future (genetic) therapeutic interventions.

Efficacy of Column Scatter Plots for Presenting Retinitis Pigmentosa Phenotypes in a Japanese Cohort.

PurposeWe evaluated the efficacy of column scatter plots to describe genotype–phenotype correlations in a Japanese cohort with retinitis pigmentosa (RP).MethodsClinical records of 121 patients with RP with identified causative mutations were reviewed. Visual acuity, central and peripheral visual fields, electroretinography (ERG), lens status, and measurements of optical coherence tomography were evaluated according to causative genes using column scatter plots. Values for three common genes (EYS, USH2A, and RHO) were compared statistically.ResultsAll patients with PDE6B, PRPH2, and RPGR mutations, those 55 years old or younger with RP1L1 and USH2A mutations, and those 45 years old or younger with EYS and RHO mutations retained visual acuity of at least 0.1. All patients with RPGR mutations showed at least −20 dB mean deviation. Goldmann perimeter measures of 4/6 patients with RHO mutations showed remaining peripheral visual fields. Dark-adapted 0.01 and 3.0 ERGs were extinguished for most genes. Half of the patients with RHO RP maintained cone responses in light-adapted 3.0 and 3.0 flicker ERG. All patients with PRPH2, those 55 years old or younger with USH2A and RP1L1, and those 45 years old or younger with PDE6B and EYS mutations maintained subfoveal ellipsoid zones. No differences were identified between EYS and USH2A or RHO and USH2A.ConclusionsColumn scatter plots enabled comparisons of the associated severities and illustration of the ophthalmological measurements for every RP causative gene.Translational RelevanceAnalysis of mutations in specific genes may be helpful for determining visual prognoses in the clinical setting.

Visual Prognosis in USH2A-Associated Retinitis Pigmentosa Is Worse for Patients with Usher Syndrome Type IIa Than for Those with Nonsyndromic Retinitis Pigmentosa

USH2A mutations are an important cause of retinitis pigmentosa (RP) with or without congenital sensorineural hearing impairment. We studied genotype-phenotype correlations and compared visual prognosis in Usher syndrome type IIa and nonsyndromic RP. Clinic-based, longitudinal, multicenter study. Consecutive patients with Usher syndrome type IIa (n= 152) and nonsyndromic RP (n= 73) resulting from USH2A mutations from ophthalmogenetic clinics in the Netherlands and Belgium. Data on clinical characteristics, visual acuity, visual field measurements, retinal imaging, and electrophysiologic features were extracted from medical charts over a mean follow-up of 9 years. Cumulative lifetime risks of low vision and blindness were estimated using Kaplan-Meier survival analysis. Low vision and blindness. Participant groups had similar distributions of gender (48% vs. 45% males in Usher syndrome type IIa vs. nonsydromic RP; P= 0.8), ethnicity (97% vs. 99% European; P= 0.3), and median follow-up time (6.5years vs. 3 years; P=0.3). Usher syndrome type IIa patients demonstrated symptoms at a younger age (median age, 15 years vs. 25 years; P < 0.001), were diagnosed earlier (median age, 26 years vs. 36.5 years; P < 0.001), and became visually impaired 13 years earlier (median age, 41 years vs. 54 years; P < 0.001) based on VF and 18 years earlier based on VA (median age, 54 years vs. 72 years; P < 0.001) than nonsyndromic RP patients. The presence of 2 truncating mutations in USH2A was associated mostly with the syndromic phenotype, whereas other combinations were present in both groups. We found novel variants in Usher syndrome type IIa (25%) and nonsyndromic RP (19%): 29 missense mutations, 10 indels, 14 nonsense mutations, 9 frameshift mutations, and 5 splice-site mutations. Most patients with USH2A-associated RP have severe visual impairment by age 50. However, those with Usher syndrome type IIa have an earlier decline of visual function and a higher cumulative risk of visualimpairment than those without nonsyndromic RP. Complete loss of function of the USH2A protein predisposes toUsher syndrome type IIa, but remnant protein function can lead to RP with or without hearing loss.

Whole Exome Sequencing Reveals Clonal Evolution of Myeloproliferative Neoplasms to Acute Myeloid Leukemia

Disease progression to acute myeloid leukemia (AML) is observed in 7% of the cases with the three classical BCR-ABL1 negative myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). According to the WHO, the presence of ≥ 20% blasts in bone marrow or peripheral blood is the diagnostic criterion for establishing leukemic transformation. PMF patients are at the highest risk, while PV and ET patients often develop secondary myelofibrosis prior to the leukemic transformation. Post-MPN AML patients have poor prognosis and clonal evolution of MPN chronic phase to AML is not well understood. Here we aimed to study the clonal evolution from MPN to AML in 7 cases, by performing whole exome sequencing (WES) on samples taken at various disease stages from individual patients.From the 7 post-MPN AML patients included in the study, 4 were diagnosed with PV and 3 with ET during chronic phase of the disease. For all 7 patients WES was performed on DNA samples from the control tissue, chronic phase and/or accelerated phase, and the leukemic phase of the disease. All variants identified by WES were validated using Sanger sequencing. In addition, tumor samples were analyzed for genomic deletions, gains and uniparental disomies (UPD) using SNP microarrays.We identified on average 16 somatic mutations (range 12-27) and 4 chromosomal aberrations (range 0-9) per patient in the leukemic stage of the disease. All patients were JAK2-V617F positive. 115 validated somatic mutations affected a total of 100 different genes. Most mutations were found in genes that were previously not linked to myeloid cancers, however, they were not recurrent. Besides JAK2-V617F, recurrent mutations were found in TP53 (N=3/7), RUNX1 (N=2/7), TET2 (N=2/7) and MPL (N=2/7). Biallelic TP53, RUNX1 and TET2 mutations were present in single patients. Known MPN and AML-related genes such as DNMT3A, SRSF2, U2AF1, IDH2, KIT, and PHF6 were mutated in single patients. We identified 25 chromosomal aberrations in 7 patients. Del5q, del6p, del7q and 9pUPD were recurrent. UPDs and trisomies of chromosomes 9, 12q, 17p and 21 were coupled with mutations in JAK2, SH2B3, TP53 and RUNX1. One patient harbored focal deletions of <1Mb on chromosomes 10 and 12, targeting TET1 and ETV6, respectively.We used variant frequencies detected by WES and copy number ratios and allelic difference values detected by microarrays at various stages of the disease to reconstruct the clonal evolution from chronic phase to AML in the 7 studied cases. Mutations with similar variant frequencies showing changes of allelic frequency in the same direction were assumed to be part of the same clone. Figure 1 illustrates an example of the proposed model for clonal evolution in Patient 6. As in this patient we had WES data from chronic, accelerated and leukemic stage of the disease, we first analyzed the clonal evolution from chronic to the accelerated phase. In the chronic phase the ~60-80% of granulocytes were derived from a single clone carrying 5 somatic mutations (JAK2, CAD, PPFIA2, SCNG, USH2A) and 2 chromosomal aberrations (1q gain and trisomy 9). At the accelerated phase of the disease we could observe that the main clone acquired somatic mutations in ADIPOQ, EYA3 and FAM123C and that there is at least one subclone (~40% of cells) appearing with mutations in OGDH, PHF6, USH2A, del7q and 9qUPD. At the leukemic stage, the clone with 9qUPD was suppressed by the outgrowing clone carrying OGDH and other mutations. We could also show that at the final leukemic stage the dominant clone acquired RUNX1 S400X mutation, amplified with a trisomy of chromosome 21, while the other RUNX1 allele mutated to Q262X.In each of the 7 studied patients the clonal evolution was unique and complex process with a few common features. Loss of TP53 is the most common genetic lesion. TET2 mutations are early events in clonal evolution of MPN and often precede the acquisition of JAK2-V617F, while RUNX1 mutations seem to be late events, leading to differentiation arrest and appearance of blasts. We demonstrated that in 6/7 studied cases the clonal evolution was a linear process, led by sequential acquisition of somatic mutations on the basis of the same clone causing the chronic disease. This finding is in line with the results of our previous study where we showed that the genetic basis of secondary AML is significantly different from de novo AML. [Display omitted] DisclosuresKralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Whole USH2A Gene Sequencing Identifies Several New Deep Intronic Mutations.

Deep intronic mutations leading to pseudoexon (PE) insertions are underestimated and most of these splicing alterations have been identified by transcript analysis, for instance, the first deep intronic mutation in USH2A, the gene most frequently involved in Usher syndrome type II (USH2). Unfortunately, analyzing USH2A transcripts is challenging and for 1.8%-19% of USH2 individuals carrying a single USH2A recessive mutation, a second mutation is yet to be identified. We have developed and validated a DNA next-generation sequencing approach to identify deep intronic variants in USH2A and evaluated their consequences on splicing. Three distinct novel deep intronic mutations have been identified. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. We present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. Moreover, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Finally, an antisense morpholino oligonucleotide tested in vitro for its ability to restore splicing caused by the c.9959-4159A>G mutation provided high inhibition rates, which are indicative of its potential for molecular therapy.

Targeted exome sequencing reveals novel USH2A mutations in Chinese patients with simplex Usher syndrome

BackgroundUsher syndrome (USH) is an autosomal recessive disorder characterized by hearing impairment and vision dysfunction due to retinitis pigmentosa. Phenotypic and genetic heterogeneities of this disease make it impractical to obtain a genetic diagnosis by conventional Sanger sequencing.MethodsIn this study, we applied a next-generation sequencing approach to detect genetic abnormalities in patients with USH. Two unrelated Chinese families were recruited, consisting of two USH afflicted patients and four unaffected relatives. We selected 199 genes related to inherited retinal diseases as targets for deep exome sequencing. Through systematic data analysis using an established bioinformatics pipeline, all variants that passed filter criteria were validated by Sanger sequencing and co-segregation analysis.ResultsA homozygous frameshift mutation (c.4382delA, p.T1462Lfs*2) was revealed in exon20 of gene USH2A in the F1 family. Two compound heterozygous mutations, IVS47 + 1G > A and c.13156A > T (p.I4386F), located in intron 48 and exon 63 respectively, of USH2A, were identified as causative mutations for the F2 family. Of note, the missense mutation c.13156A > T has not been reported so far.ConclusionIn conclusion, targeted exome sequencing precisely and rapidly identified the genetic defects in two Chinese USH families and this technique can be applied as a routine examination for these disorders with significant clinical and genetic heterogeneity.

Whole-exome sequencing identifies USH2A mutations in a pseudo-dominant Usher syndrome family.

Usher syndrome (USH) is an autosomal recessive (AR) multi-sensory degenerative disorder leading to deaf-blindness. USH is clinically subdivided into three subclasses, and 10 genes have been identified thus far. Clinical and genetic heterogeneities in USH make a precise diagnosis difficult. A dominant-like USH family in successive generations was identified, and the present study aimed to determine the genetic predisposition of this family. Whole-exome sequencing was performed in two affected patients and an unaffected relative. Systematic data were analyzed by bioinformatic analysis to remove the candidate mutations via step-wise filtering. Direct Sanger sequencing and co-segregation analysis were performed in the pedigree. One novel and two known mutations in the USH2A gene were identified, and were further confirmed by direct sequencing and co-segregation analysis. The affected mother carried compound mutations in the USH2A gene, while the unaffected father carried a heterozygous mutation. The present study demonstrates that whole-exome sequencing is a robust approach for the molecular diagnosis of disorders with high levels of genetic heterogeneity.

Next generation sequencing based molecular genetic analysis of a Chinese Han family with autosomal retinitis pigmentosa

Background Retinitis pigmeutosa (RP) is a progressive inheritance disease. It is characterized by highly genetical and phenotypical heterogeneity. With the rapid development of genomics, new methods are applied to the genetic screening of RP. Objective This study was to characterize the clinical features of a Chinese family with autosomal RP and to screen the candidate genes. Methods Twelve members from this family were included in the study. All participants underwent complete ophthalmologic examinations. Targeted-capture next generation sequencing (NGS) based molecular genetic analysis was performed on two patients of this RP family(Ⅱ5, Ⅱ7). The DNA sample from the two patients was separately sequenced using custom capture gene chip, which includes 59 retinal disease genes. The sequencing results were analyzed by bioinformatics technology. Identified variations were verified in the rest family members by PCR and Sanger sequencing. This study was approved by Ethic Committee of West China Hospital, and informed consent was obtained from the subjects. Results Four members of this family were diagnosed as RP, and the rest were asymptomatic. Missense mutation (c. 3065T>C, p. Phe1022Ser) in USH2A and missense mutation (c. 1699G>A, p. Ala1319Gly) in PDE6A were found in two patients (Ⅱ5 and Ⅱ7). The variants were not co-segregated with the phenotype of this family. The causative mutation was not found by the targeted-capture NGS based eye disease chip, but it ruled out a large number of candidate genes for RP. Conclusions Our study suggests that targeted-capture NGS based eye disease chip can quickly detect mutations in known RP genes. It can be a new applicable and efficient method for molecular genetic analysis of ocular disease. Key words: Retinitis pigmentosa; Gene; Next generation sequencing; Molecular genetic analysis; Pedigree