Exome sequencing reveals a homozygous SYT14 mutation in adult-onset, autosomal-recessive spinocerebellar ataxia with psychomotor retardation

Autosomal-recessive cerebellar ataxias (ARCAs) are clinically and genetically heterogeneous disorders associated with diverse neurological and nonneurological features that occur before the age of 20. Currently, mutations in more than 20 genes have been identified, but approximately half of the ARCA patients remain genetically unresolved. In this report, we describe a Japanese family in which two siblings have slow progression of a type of ARCA with psychomotor retardation. Using whole-exome sequencing combined with homozygosity mapping, we identified a homozygous missense mutation in SYT14, encoding synaptotagmin XIV (SYT14). Expression analysis of the mRNA of SYT14 by a TaqMan assay confirmed that SYT14 mRNA was highly expressed in human fetal and adult brain tissue as well as in the mouse brain (especially in the cerebellum). In an in vitro overexpression system, the mutant SYT14 showed intracellular localization different from that of the wild-type. An immunohistochemical analysis clearly showed that SYT14 is specifically localized to Purkinje cells of the cerebellum in humans and mice. Synaptotagmins are associated with exocytosis of secretory vesicles (including synaptic vesicles), indicating that the alteration of the membrane-trafficking machinery by the SYT14 mutation may represent a distinct pathomechanism associated with human neurodegenerative disorders.     

Targeted next-generation sequencing identifies ABCA4 mutations in Chinese families with childhood-onset and adult-onset Stargardt disease

Background: Stargardt disease (STGD) is the most common form of juvenile macular dystrophy associated with progressive central vision loss, and is agenetically and clinically heterogeneous disease. Molecular diagnosis is of great significance in aiding the clinical diagnosis, helping to determine the phenotypic severity and visual prognosis. In the present study, we determined the clinical and genetic features of seven childhood-onset and three adult-onset Chinese STGD families. We performed capture next-generation sequencing (NGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes.Methods: In all, ten unrelated Chinese families were enrolled. Panel-based NGS was performed to identify potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes, including the five known STGD genes (ABCA4, PROM1, PRPH2, VMD2, and ELOVL4). Variant analysis, Sanger validation, and segregation tests were utilized to validate the disease-causing mutations in these families.Results: Using systematic data analysis with an established bioinformatics pipeline and segregation analysis, 17 pathogenic mutations in ABCA4 were identified in the 10 STGD families. Four of these mutations were novel: c.371delG, c.681T > G, c.5509C > T, and EX37del. Childhood-onset STGD was associated with severe visual loss, generalized retinal dysfunction and was due to more severe variants in ABCA4 than those found in adult-onset disease.Conclusions: We expand the existing spectrum of STGD and reveal the genotype–phenotype relationships of the ABCA4 mutations in Chinese patients. Childhood-onset STGD lies at the severe end of the spectrum of ABCA4-associated retinal phenotypes.     

Targeted next generation sequencing identifies clinically actionable mutations in patients with melanoma

Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here, we describe a NextGen sequencing approach to fully analyzing 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high‐throughput sequencing and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard‐of‐care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma.     

Targeted next-generation sequencing identifies a homozygous nonsense mutation in ABHD12, the gene underlying PHARC, in a family clinically diagnosed with Usher syndrome type 3

ABSTRACT: BACKGROUND: Usher syndrome (USH) is an autosomal recessive genetically heterogeneous disorder with congenital sensorineural hearing impairment and retinitis pigmentosa (RP). We have identified a consanguineous Lebanese family with two affected members displaying progressive hearing loss, RP and cataracts, therefore clinically diagnosed as USH type 3 (USH3). Our study was aimed at the identification of the causative mutation in this USH3-like family. METHODS: Candidate loci were identified using genomewide SNP-array-based homozygosity mapping followed by targeted enrichment and next-generation sequencing. RESULTS: Using a capture array targeting the three identified homozygosity-by-descent regions on chromosomes 1q43-q44, 20p13-p12.2 and 20p11.23-q12, we identified a homozygous nonsense mutation, p.Arg65X, in ABHD12 segregating with the phenotype. CONCLUSION: Mutations of ABHD12, an enzyme hydrolyzing an endocannabinoid lipid transmitter, cause PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract). After the identification of the ABHD12 mutation in this family, one patient underwent neurological examination which revealed ataxia, but no polyneuropathy. ABHD12 is not known to be related to the USH protein interactome. The phenotype of our patient represents a variant of PHARC, an entity that should be taken into account as differential diagnosis for USH3. Our study demonstrates the potential of comprehensive genetic analysis for improving the clinical diagnosis.     

Targeted sequencing of the 9p21.3 region reveals association with reduced disease risks in Ashkenazi Jewish centenarians

Genome-wide association studies (GWAS) have pinpointed the chromosomal locus 9p21.3 as a genetic hotspot for various age-related disorders. Common genetic variants in this locus are linked to multiple traits, including coronary artery diseases, cancers, and diabetes. Centenarians are known for their reduced risk and delayed onset of these conditions. To investigate whether this evasion of disease risks involves diminished genetic risks in the 9p21.3 locus, we sequenced this region in an Ashkenazi Jewish centenarian cohort (centenarians: n = 450, healthy controls: n = 500). Risk alleles associated with cancers, glaucoma, CAD, and T2D showed a significant depletion in centenarians. Furthermore, the risk and non-risk genotypes are linked to two distinct low-frequency variant profiles, enriched in controls and centenarians, respectively. Our findings provide evidence that the extreme longevity cohort is associated with collectively lower risks of multiple age-related diseases in the 9p21.3 locus.     

Application of next-generation sequencing to screen for pathogenic mutations in 123 unrelated Chinese patients with Marfan syndrome or a related disease

Marfan syndrome(MFS) is a systemic connective tissue disease principally affecting the ocular, skeletal and cardiovascular systems. This autosomal dominant disorder carries a prevalence of 1:3,000 to 1:5,000. This study aims to define the mutational spectrum of MFS related genes in Chinese patients and to establish genotype-phenotype correlations in MFS. Panel-based targeted next-generation sequencing was used to analyze the FBN1, TGFBR1 and TGFBR2 genes in 123 unrelated Chinese individuals with MFS or a related disease. Genotype-phenotype correlation analyses were performed in mutation-positive patients. The results showed that 97 cases/families(78.9%; 97/123) harbor at least one(likely) pathogenic mutation, most of which were in FBN1; four patients had TGFBR1/2 mutations; and one patient harbored a SMAD3 mutation. Three patients had two FBN1 mutations, and all patients showed classical MFS phenotypes. Patients with a dominant negative-FBN1 mutation had a higher prevalence of ectopia lentis(EL). Patients carrying a haploinsufficiency-FBN1 mutation tended to have aortic dissection without EL. This study extends the spectrum of genetic backgrounds of MFS and enriches our knowledge of genotype-phenotype correlations.     

Targeted next generation sequencing identifies novel NOTCH3 gene mutations in CADASIL diagnostics patients

Background

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic, hereditary, small vessel disease of the brain causing stroke and vascular dementia in adults. CADASIL has previously been shown to be caused by varying mutations in the NOTCH3 gene. The disorder is often misdiagnosed due to its significant clinical heterogeneic manifestation with familial hemiplegic migraine and several ataxia disorders as well as the location of the currently identified causative mutations. The aim of this study was to develop a new, comprehensive and efficient single assay strategy for complete molecular diagnosis of NOTCH3 mutations through the use of a custom next-generation sequencing (NGS) panel for improved routine clinical molecular diagnostic testing.

Results

Our custom NGS panel identified nine genetic variants in NOTCH3 (p.D139V, p.C183R, p.R332C, p.Y465C, p.C597W, p.R607H, p.E813E, p.C977G and p.Y1106C). Six mutations were stereotypical CADASIL mutations leading to an odd number of cysteine residues in one of the 34 NOTCH3 gene epidermal growth factor (EGF)-like repeats, including three new typical cysteine mutations identified in exon 11 (p.C597W; c.1791C>G); exon 18 (p.C977G; c.2929T>G) and exon 20 (p.Y1106C; c.3317A>G). Interestingly, a novel missense mutation in the CACNA1A gene was also identified in one CADASIL patient. All variants identified (novel and known) were further investigated using in silico bioinformatic analyses and confirmed through Sanger sequencing.

Conclusions

NGS provides an improved and effective methodology for the diagnosis of CADASIL. The NGS approach reduced time and cost for comprehensive genetic diagnosis, placing genetic diagnostic testing within reach of more patients.

     

Exome sequencing reveals VCP mutations as a cause of familial ALS

Using exome sequencing, we identified a p.R191Q amino acid change in the valosin-containing protein (VCP) gene in an Italian family with autosomal dominantly inherited amyotrophic lateral sclerosis (ALS). Mutations in VCP have previously been identified in?families with Inclusion Body Myopathy, Paget disease, and Frontotemporal Dementia (IBMPFD). Screening of VCP in a cohort of 210 familial ALS cases and 78 autopsy-proven ALS cases identified four additional mutations including a p.R155H mutation in a pathologically proven case of ALS. VCP protein is essential for maturation of ubiquitin-containing autophagosomes, and mutant VCP toxicity is partially mediated through its effect on TDP-43 protein, a major constituent of ubiquitin inclusions that neuropathologically characterize ALS. Our data broaden the phenotype of IBMPFD to include motor neuron degeneration, suggest that VCP mutations may account for ～1%-2% of familial ALS, and provide evidence directly implicating defects in the ubiquitination/protein degradation pathway in motor neuron degeneration.     

Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis

Background

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive.

Results

Here we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified.

Conclusions

Using this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1263-4) contains supplementary material, which is available to authorized users.     

Targeted/exome sequencing identified mutations in ten Chinese patients diagnosed with Noonan syndrome and related disorders

Background

Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML) are autosomal dominant developmental disorders. NS and NSML are caused by abnormalities in genes that encode proteins related to the RAS-MAPK pathway, including PTPN11, RAF1, BRAF, and MAP2K. In this study, we diagnosed ten NS or NSML patients via targeted sequencing or whole exome sequencing (TS/WES).

Methods

TS/WES was performed to identify mutations in ten Chinese patients who exhibited the following manifestations: potential facial dysmorphisms, short stature, congenital heart defects, and developmental delay. Sanger sequencing was used to confirm the suspected pathological variants in the patients and their family members.

Results

TS/WES revealed three mutations in the PTPN11 gene, three mutations in RAF1 gene, and four mutations in BRAF gene in the NS and NSML patients who were previously diagnosed based on the abovementioned clinical features. All the identified mutations were determined to be de novo mutations. However, two patients who carried the same mutation in the RAF1 gene presented different clinical features. One patient with multiple lentigines was diagnosed with NSML, while the other patient without lentigines was diagnosed with NS. In addition, a patient who carried a hotspot mutation in the BRAF gene was diagnosed with NS instead of cardiofaciocutaneous syndrome (CFCS).

Conclusions

TS/WES has emerged as a useful tool for definitive diagnosis and accurate genetic counseling of atypical cases. In this study, we analyzed ten Chinese patients diagnosed with NS and related disorders and identified their correspondingPTPN11, RAF1, and BRAF mutations. Among the target genes, BRAF showed the same degree of correlation with NS incidence as that of PTPN11 or RAF1.

     

Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome

Olmsted syndrome (OS) is a rare congenital disorder characterized by palmoplantar and periorificial keratoderma, alopecia in most cases, and severe itching. The genetic basis for OS remained unidentified. Using whole-exome sequencing of case-parents trios, we have identified a de novo missense mutation in TRPV3 that produces p.Gly573Ser in an individual with OS. Nucleotide sequencing of five additional affected individuals also revealed missense mutations in TRPV3 (which produced p.Gly573Ser in three cases and p.Gly573Cys and p.Trp692Gly in one case each). Encoding a transient receptor potential vanilloid-3 cation channel, TRPV3 is primarily expressed in the skin, hair follicles, brain, and spinal cord. In transfected HEK293 cells expressing TRPV3 mutants, much larger inward currents were recorded, probably because of the constitutive opening of the mutants. These gain-of-function mutations might lead to elevated apoptosis of keratinocytes and consequent skin hyperkeratosis in the affected individuals. Our findings suggest that TRPV3 plays essential roles in skin keratinization, hair growth, and possibly itching sensation in humans and selectively targeting TRPV3 could provide therapeutic potential for keratinization or itching-related skin disorders.     

Exome sequencing reveals a novel TTC19 mutation in an autosomal recessive spinocerebellar ataxia patient

Background

Genetic modifiers are important clues for the identification of therapeutic targets in neurodegenerative diseases. Huntington disease (HD) is one of the most common autosomal dominant inherited neurodegenerative diseases. The clinical symptoms include motor abnormalities, cognitive decline and behavioral disturbances. Symptom onset is typically between 40 and 50 years of age, but can vary by several decades in extreme cases and this is in part determined by modifying genetic factors. The metabolic master regulator PGC-1α, coded by the PPARGC1A gene, coordinates cellular respiration and was shown to play a role in neurodegenerative diseases, including HD.

Methods

Using a candidate gene approach we analyzed a large European cohort (n?=?1706) from the REGISTRY study for associations between PPARGC1A genotype and age at onset (AO) in HD.

Results

We report that a coding variant (rs3736265) in PPARGC1A is associated with an earlier motor AO in men but not women carrying the HD mutation.

Conclusions

These results further strengthen the evidence for a role of PGC-1α in HD and unexpectedly suggest a gender effect.     

Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia

Polyglutamine disorders are inherited neurodegenerative diseases caused by the accumulation of expanded polyglutamine protein (polyQ). Previously, we identified a new guanosine triphosphatase, CRAG, which facilitates the degradation of polyQ aggregates through the ubiquitin-proteasome pathway in cultured cells. Because expression of CRAG decreases in the adult brain, a reduced level of CRAG could underlie the onset of polyglutamine diseases. To examine the potential of CRAG expression for treating polyglutamine diseases, we generated model mice expressing polyQ predominantly in Purkinje cells. The model mice showed poor dendritic arborization of Purkinje cells, a markedly atrophied cerebellum and severe ataxia. Lentivector-mediated expression of CRAG in Purkinje cells of model mice extensively cleared polyQ aggregates and re-activated dendritic differentiation, resulting in a striking rescue from ataxia. Our in vivo data substantiate previous cell-culture-based results and extend further the usefulness of targeted delivery of CRAG as a gene therapy for polyglutamine diseases.