Pediatric Myelodysplastic Syndrome With Germline RRAS Mutation: Expanding the Phenotype of RASopathies.

The RAS/mitogen-activated protein kinase pathway plays a significant role in cell cycle regulation. Germline mutation of this pathway leads to overlapping genetic disorders, RASopathies, and is also an important component of tumorigenesis. Here we describe a rare case of myelodysplastic syndrome with monosomy 7 in a pediatric patient with a germline RRAS mutation. RRAS mutations have been implicated in the development of juvenile myelomonocytic leukemia, but our case suggests RRAS mutations display a broader malignant potential. Our case supports the recommendation that genetic testing should include RRAS in suspected RASopathy patients and if identified, these patients undergo surveillance for hematologic malignancy.

Functional variants in TBX2 are associated with a syndromic cardiovascular and skeletal developmental disorder.

The 17 genes of the T-box family are transcriptional regulators that are involved in all stages of embryonic development, including craniofacial, brain, heart, skeleton and immune system. Malformation syndromes have been linked to many of the T-box genes. For example, haploinsufficiency of TBX1 is responsible for many structural malformations in DiGeorge syndrome caused by a chromosome 22q11.2 deletion. We report four individuals with an overlapping spectrum of craniofacial dysmorphisms, cardiac anomalies, skeletal malformations, immune deficiency, endocrine abnormalities and developmental impairments, reminiscent of DiGeorge syndrome, who are heterozygotes for TBX2 variants. The p.R20Q variant is shared by three affected family members in an autosomal dominant manner; the fourth unrelated individual has a de novo p.R305H mutation. Bioinformatics analyses indicate that these variants are rare and predict them to be damaging. In vitro transcriptional assays in cultured cells show that both variants result in reduced transcriptional repressor activity of TBX2. We also show that the variants result in reduced protein levels of TBX2. Heterologous over-expression studies in Drosophila demonstrate that both p.R20Q and p.R305H function as partial loss-of-function alleles. Hence, these and other data suggest that TBX2 is a novel candidate gene for a new multisystem malformation disorder.

Functional Dysregulation of CDC42 Causes Diverse Developmental Phenotypes.

Exome sequencing has markedly enhanced the discovery of genes implicated in Mendelian disorders, particularly for individuals in whom a known clinical entity could not be assigned. This has led to the recognition that phenotypic heterogeneity resulting from allelic mutations occurs more commonly than previously appreciated. Here, we report that missense variants in CDC42, a gene encoding a small GTPase functioning as an intracellular signaling node, underlie a clinically heterogeneous group of phenotypes characterized by variable growth dysregulation, facial dysmorphism, and neurodevelopmental, immunological, and hematological anomalies, including a phenotype resembling Noonan syndrome, a developmental disorder caused by dysregulated RAS signaling. In silico, in vitro, and in vivo analyses demonstrate that mutations variably perturb CDC42 function by altering the switch between the active and inactive states of the GTPase and/or affecting CDC42 interaction with effectors, and differentially disturb cellular and developmental processes. These findings reveal the remarkably variable impact that dominantly acting CDC42 mutations have on cell function and development, creating challenges in syndrome definition, and exemplify the importance of functional profiling for syndrome recognition and delineation.

Correction: Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases.

In the published version of this paper, some of the columns in the last three rows of Table 3 were mistakenly transposed. The corrected table appears below. In col. 6 of the row for DNMT3A, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S1". In col. 6 of the row for SON, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S2".

Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases.

PURPOSE: Diagnostic exome sequencing (DES) is now a commonly ordered test for individuals with undiagnosed genetic disorders. In addition to providing a diagnosis for characterized diseases, exome sequencing has the capacity to uncover novel candidate genes for disease. METHODS: Family-based DES included analysis of both characterized and novel genetic etiologies. To evaluate candidate genes for disease in the clinical setting, we developed a systematic, rule-based classification schema. RESULTS: Testing identified a candidate gene among 7.7% (72/934) of patients referred for DES; 37 (4.0%) and 35 (3.7%) of the genes received evidence scores of "candidate" and "suspected candidate," respectively. A total of 71 independent candidate genes were reported among the 72 patients, and 38% (27/71) were subsequently corroborated in the peer-reviewed literature. This rate of corroboration increased to 51.9% (27/52) among patients whose gene was reported at least 12 months previously. CONCLUSIONS: Herein, we provide transparent, comprehensive, and standardized scoring criteria for the clinical reporting of candidate genes. These results demonstrate that DES is an integral tool for genetic diagnosis, especially for elucidating the molecular basis for both characterized and novel candidate genetic etiologies. Gene discoveries also advance the understanding of normal human biology and more common diseases.Genet Med 19 2, 224-235.

CLTC as a clinically novel gene associated with multiple malformations and developmental delay.

Diagnostic exome sequencing has recently emerged as an invaluable tool in determining the molecular etiology of cases involving dysmorphism and developmental delay that are otherwise unexplained by more traditional methods of genetic testing. Our patient was large for gestational age at 35 weeks, delivered to a 27-year-old primigravid Caucasian whose pregnancy was complicated by preeclampsia. Neonatal period was notable for hypoglycemia, apnea, bradycardia, hyperbilirubinemia, grade I intraventricular hemorrhage, subdural hematoma, laryngomalacia, hypotonia, and feeding difficulties. The patient had numerous minor dysmorphic features. At three and a half years of age, she has global developmental delays and nystagmus, and is being followed for a mediastinal neuroblastoma that is currently in remission. Karyotype and oligo-microarray were normal. Whole-exome, next generation sequencing (NGS) coupled to bioinformatic filtering and expert medical review at Ambry Genetics revealed 14 mutations in 9 genes, and these genes underwent medical review. A heterozygous de novo frameshift mutation, c.2737_2738dupGA p.D913Efs*59, in which two nucleotides are duplicated in exon 17 of the CLTC gene, results in substitution of glutamic acid for aspartic acid at position 913 of the protein, as well as a frame shift that results in a premature termination codon situated 58 amino acids downstream. Clathrin Heavy Chain 1 (CHC1) has been shown to play an important role in the brain for vesicle recycling and neurotransmitter release at pre-synaptic nerve terminals. There is also evidence implicating it in the proper development of the placenta during the early stages of pregnancy. The CLTC alteration identified herein is likely to provide an explanation for the patient's adverse phenotype. Ongoing functional studies will further define the impact of this alteration on CHC1 function and consequently, human disease.

Diagnostic exome sequencing provides a molecular diagnosis for a significant proportion of patients with epilepsy.

PURPOSE: To assess the yield of diagnostic exome sequencing (DES) and to characterize the molecular findings in characterized and novel disease genes in patients with epilepsy. METHODS: In an unselected sample of 1,131 patients referred for DES, overall results were compared between patients with and without epilepsy. DES results were examined based on age of onset and epilepsy diagnosis. RESULTS: Positive/likely positive results were identified in 112/293 (38.2%) epilepsy patients compared with 210/732 (28.7%) patients without epilepsy (P = 0.004). The diagnostic yield in characterized disease genes among patients with epilepsy was 33.4% (105/314). KCNQ2, MECP2, FOXG1, IQSEC2, KMT2A, and STXBP1 were most commonly affected by de novo alterations. Patients with epileptic encephalopathies had the highest rate of positive findings (43.4%). A likely positive novel genetic etiology was proposed in 14/200 (7%) patients with epilepsy; this frequency was highest in patients with epileptic encephalopathies (17%). Three genes (COQ4, DNM1, and PURA) were initially reported as likely positive novel disease genes and were subsequently corroborated in independent peer-reviewed publications. CONCLUSION: DES with analysis and interpretation of both characterized and novel genetic etiologies is a useful diagnostic tool in epilepsy, particularly in severe early-onset epilepsy. The reporting on novel genetic etiologies may further increase the diagnostic yield.Genet Med 18 9, 898-905.

Diagnostic Exome Sequencing Identifies a Novel Gene, EMILIN1, Associated with Autosomal-Dominant Hereditary Connective Tissue Disease.

Heritable connective tissue diseases are a highly heterogeneous family of over 200 disorders that affect the extracellular matrix. While the genetic basis of several disorders is established, the etiology has not been discovered for a large portion of patients, likely due to rare yet undiscovered disease genes. By performing trio-exome sequencing of a 55-year-old male proband presenting with multiple symptoms indicative of a connective disorder, we identified a heterozygous missense alteration in exon 1 of the Elastin Microfibril Interfacer 1 (EMILIN1) gene, c.64G>A (p.A22T). The proband presented with ascending and descending aortic aneurysms, bilateral lower leg and foot sensorimotor peripheral neuropathy, arthropathy, and increased skin elasticity. Sanger sequencing confirmed that the EMILIN1 alteration, which maps around the signal peptide cleavage site, segregated with disease in the affected proband, mother, and son. The impaired secretion of EMILIN-1 in cells transfected with the mutant p.A22T coincided with abnormal protein accumulation within the endoplasmic reticulum. In skin biopsy of the proband, we detected less EMILIN-1 with disorganized and abnormal coarse fibrils, aggregated deposits underneath the epidermis basal lamina, and dermal cells apoptosis. These findings collectively suggest that EMILIN1 may represent a new disease gene associated with an autosomal-dominant connective tissue disorder.

Germline activating MTOR mutation arising through gonadal mosaicism in two brothers with megalencephaly and neurodevelopmental abnormalities.

BACKGROUND: In humans, Mammalian Target of Rapamycin (MTOR) encodes a 300 kDa serine/ threonine protein kinase that is ubiquitously expressed, particularly at high levels in brain. MTOR functions as an integrator of multiple cellular processes, and in so doing either directly or indirectly regulates the phosphorylation of at least 800 proteins. While somatic MTOR mutations have been recognized in tumors for many years, and more recently in hemimegalencephaly, germline MTOR mutations have rarely been described. CASE PRESENTATION: We report the successful application of family-trio Diagnostic Exome Sequencing (DES) to identify the underlying molecular etiology in two brothers with multiple neurological and developmental lesions, and for whom previous testing was non-diagnostic. The affected brothers, who were 6 and 23 years of age at the time of DES, presented symptoms including but not limited to mild Autism Spectrum Disorder (ASD), megalencephaly, gross motor skill delay, cryptorchidism and bilateral iris coloboma. Importantly, we determined that each affected brother harbored the MTOR missense alteration p.E1799K (c.5395G>A). This exact variant has been previously identified in multiple independent human somatic cancer samples and has been shown to result in increased MTOR activation. Further, recent independent reports describe two unrelated families in whom p.E1799K co-segregated with megalencephaly and intellectual disability (ID); in both cases, p.E1799K was shown to have originated due to germline mosaicism. In the case of the family reported herein, the absence of p.E1799K in genomic DNA extracted from the blood of either parent suggests that this alteration most likely arose due to gonadal mosaicism. Further, the p.E1799K variant exerts its effect by a gain-of-function (GOF), autosomal dominant mechanism. CONCLUSION: Herein, we describe the use of DES to uncover an activating MTOR missense alteration of gonadal mosaic origin that is likely to be the causative mutation in two brothers who present multiple neurological and developmental abnormalities. Our report brings the total number of families who harbor MTOR p.E1799K in association with megalencephaly and ID to three. In each case, evidence suggests that p.E1799K arose in the affected individuals due to gonadal mosaicism. Thus, MTOR p.E1799K can now be classified as a pathogenic GOF mutation that causes megalencephaly and cognitive impairment in humans.

Enhanced utility of family-centered diagnostic exome sequencing with inheritance model-based analysis: results from 500 unselected families with undiagnosed genetic conditions.

PURPOSE: Diagnostic exome sequencing was immediately successful in diagnosing patients in whom traditional technologies were uninformative. Herein, we provide the results from the first 500 probands referred to a clinical laboratory for diagnostic exome sequencing. METHODS: Family-based exome sequencing included whole-exome sequencing followed by family inheritance-based model filtering, comprehensive medical review, familial cosegregation analysis, and analysis of novel genes. RESULTS: A positive or likely positive result in a characterized gene was identified in 30% of patients (152/500). A novel gene finding was identified in 7.5% of patients (31/416). The highest diagnostic rates were observed among patients with ataxia, multiple congenital anomalies, and epilepsy (44, 36, and 35%, respectively). Twenty-three percent of positive findings were within genes characterized within the past 2 years. The diagnostic rate was significantly higher among families undergoing a trio (37%) as compared with a singleton (21%) whole-exome testing strategy. CONCLUSION: Overall, we present results from the largest clinical cohort of diagnostic exome sequencing cases to date. These data demonstrate the utility of family-based exome sequencing and analysis to obtain the highest reported detection rate in an unselected clinical cohort, illustrating the utility of diagnostic exome sequencing as a transformative technology for the molecular diagnosis of genetic disease.

A capillary electrophoresis sequencing method for the identification of mutations in the inverted terminal repeats of adeno-associated virus.

Inverted terminal repeat (ITR) integrity is critical for the replication, packaging, and transduction of recombinant adeno-associated virus (rAAV), a promising gene therapy vector. Because AAV ITRs possess 70% GC content and are palindromic, they are notoriously difficult to sequence. The purpose of this work was to develop a reliable ITR sequencing method. The ITRs of two molecular clones of AAV2, pTZAAV and pAV2, were (1) sequenced directly from plasmid DNA in the presence of denaturant (direct sequencing method, DSM) or (2) first amplified in a reaction in which 7-deaza-dGTP was substituted for dGTP and the resultant amplification product sequenced (amplification sequencing method, ASM). The DSM and ASM techniques yielded clear chromatograms, read through the ITR hairpin, and revealed hitherto unreported mutations in each ITR. pTZAAV and pAV2 possess identical mutations at the upstream MscI site of the 5' ITR (T>G, nt 2) and the downstream MscI site of the 3' ITR (del. nt 4672-4679). The chromatograms for pAV2 also revealed that the ITRs of this construct were arranged in a FLOP/FLOP orientation. In addition, the DSM was successfully employed to recover ITR-chromosomal junction sequences from a variety of rAAV-transduced tissue types. Both the DSM and ASM can be employed to sequence through the AAV ITR hairpin, and both techniques reliably detect mutations in the ITR. Because the DSM and ASM offer a way to verify ITR integrity, they constitute powerful tools for the process development of rAAV gene therapy.

Toward a fluorescent single-strand conformation polymorphism technique that detects all mutations: F-DOVAM-S.

Although DOVAM-S (detection of virtually all mutations-SSCP) in effect detects all mutations and is less costly than direct sequencing, the technique currently requires the use of radioactivity. F-DOVAM-S (fluorescent DOVAM-S) was developed to replace the isotopic label with fluorescence and to increase throughput via dye color multiplexing. As proof of principle, two multitemperature slab gel electrophoresis conditions were evaluated through the blinded analysis of mutations in the factor IX (FIX) genes of 88 hemophilia B (HB) patients and 7 wild-type controls. Using only two conditions, it was determined that F-DOVAM-S had a detection sensitivity of 97%. It is anticipated that when three or four optimized conditions are employed, F-DOVAM-S will detect all mutations. Three patient samples were multiplexed per well using three different fluorescent dyes (6FAM, VIC, and NED), demonstrating that it is possible to analyze up to 44 kb of diploid, color-coded amplification product per gel lane. This value corresponds to a throughput of approximately 4 Mb of DNA analyzed per 96-well gel, which is approximately triple that of conventional radiolabeled DOVAM-S. Throughput is further enhanced by the rapidity at which the fluorescent signal can be captured and the resultant multicolor chromatograms analyzed. Given these data, F-DOVAM-S has the potential to be a particularly powerful technology for clinical diagnosis because it allows the mutation analysis of multiple patients to be performed within 24h.

TNF ligands: is TALL-1 a trimer or a virus-like cluster?

Native TALL-1 (B-cell activation factor, BAFF; also known as BlyS) was initially described as a homotrimer, but Liu and colleagues claim that it is a 60-subunit complex on the basis of their results from X-ray crystallography and size-exclusion chromatography. They consider TALL-1 60-mers to be the biologically active form, and the arrangement of the 60-mers resembles that of the capsid of satellite tobacco necrosis virus. Here we show that active TALL-1 is trimeric under normal physiological conditions and that formation of higher-order oligomers is an artefact of tagging the amino terminus of the protein with a histidine tag.

A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells.

In platelets, PGHS-1-dependant formation of thromboxane A(2) is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are a-nucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01 which can be induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression. Using a specific antibody to PGHS-1 we have developed a technique utilizing immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein levels as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 x 10(-8) M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen, CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation between PGHS-1 levels and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein level, which shows a consistent increase over the entire course of differentiation, can be used as an additional and better index by which to monitor megakaryocyte differentiation.