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Biallelic loss-of-function variants in the thrombospondin-type laminin G domain and epilepsy-associated repeats (TSPEAR) gene have recently been associated with ectodermal dysplasia and hearing loss. The first reports describing a TSPEAR disease association identified this gene is a cause of nonsyndromic hearing loss, but subsequent reports involving additional affected families have questioned this evidence and suggested a stronger association with ectodermal dysplasia. To clarify genotype-phenotype associations for TSPEAR variants, we characterized 13 individuals with biallelic TSPEAR variants. Individuals underwent either exome sequencing or panel-based genetic testing. Nearly all of these newly reported individuals (11/13) have phenotypes that include tooth agenesis or ectodermal dysplasia, while three newly reported individuals have hearing loss. Of the individuals displaying hearing loss, all have additional variants in other hearing-loss-associated genes, specifically TMPRSS3, GJB2, and GJB6, that present competing candidates for their hearing loss phenotype. When presented alongside previous reports, the overall evidence supports the association of TSPEAR variants with ectodermal dysplasia and tooth agenesis features but creates significant doubt as to whether TSPEAR variants are a monogenic cause of hearing loss. Further functional evidence is needed to evaluate this phenotypic association., (© 2021 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Purpose: The ability of a single technology, next-generation sequencing, to provide both sequence and copy number variant (CNV) results has driven the merger of clinical cytogenetics and molecular genetics. Consequently, the distinction between the definition of a sequence variant and a CNV is blurry. As the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) standards and guidelines for interpretation of sequence variants address CNV classification only sparingly, this study focused on adapting ACMG/AMP criteria for single-gene CNV interpretation., Methods: CNV-specific modifications of the 2015 ACMG/AMP criteria were developed and their utility was independently tested by three diagnostic laboratories. Each laboratory team interpreted the same 12 single-gene CNVs using three systems: (1) without ACMG/AMP guidance, (2) with ACMG/AMP criteria, and (3) with new modifications. A replication study of 12 different CNVs validated the modified criteria., Results: The adapted criteria system presented here showed improved concordance and usability for single-gene CNVs compared with using the ACMG/AMP interpretation guidelines focused on sequence variants., Conclusion: These single-gene CNV criteria modifications could be used as a supplement to the ACMG/AMP guidelines for sequence variants, allowing for a streamlined workflow and a step toward a uniform classification system for both sequence and copy number alterations.

Phosphoglycerate kinase-1 (PGK1) deficiency is a rare X-linked disorder caused by pathogenic variants in the PGK1 gene. Complete loss-of-function variants have not been reported in this gene, indicating that residual enzyme function is critical for viability in males. Therefore, copy number variants (CNVs) that include single exon or multiple exon deletions or duplications are generally not expected in individuals with PGK1 deficiency. Here we describe a 64-year-old male presenting with a family history (three additional affected males) and a personal history of childhood-onset metabolic myopathy that involves episodes of muscle pain, stiffness after activity, exercise intolerance, and myoglobinuria after exertion. Biochemical analysis on a muscle biopsy indicated significantly reduced activity (15% compared to normal) for phosphoglycerate kinase (PGK1), a glycolytic enzyme encoded by PGK1. A diagnosis of PGK1 deficiency was established by molecular analysis which detected an approximately 886 kb deletion involving the polyadenylation site in the 3'UTR of the PGK1 gene. RNA analysis showed significantly reduced PGK1 transcript levels (30% compared to normal). This is the first deletion reported in the PGK1 gene and is the first pathogenic variant involving the 3'UTR polyadenylation site of this gene. Our report emphasizes the role of 3'UTR variants in human disorders and underscores the need for exploring noncoding regions of disease-associated genes when seeking a molecular diagnosis.

Background: Patient satisfaction to Antenatal care services has traditionally been linked to the quality of services given and the extent to which specific needs are met. Even though data in this area was limited in Ethiopia, improving quality of care was one of the strategies in health sector development program IV. This study, therefore, attempted to assess client satisfaction to skilled antenatal care services in the study area., Methods and Materials: A cross-sectional facility based survey was conducted among women who were attending antenatal care clinic, using quantitative method triangulated with qualitative data collection. Participants were selected using systematic sampling method according to the flow pregnant women to the antenatal care clinics. The study was carried out in all functional public health centers in the district. During the survey, 405 women were interviewed. A logistic regression model was applied to control for confounders., Results: Out of the total respondents, overall satisfied to skilled antenatal care services were about 277(68%). The most common specific component of antenatal care that had good-satisfaction by the respondents was "Privacy" at examination (81.7%). Most satisfied health education session was "Diet and nutrition" session (82.2%). Absence of sonar test, no doctor and long waiting time were commonest causes of dissatisfaction. Respondents who have > 2 previous antenatal care visit were 3 times more likely (AOR = 2.93; 95% CI, 1.21, 7.12) to have satisfaction to antenatal care services as compared to those with < 1 visit. Women whose current visit fourth were 9 times more likely (AOR = 9.02, 95% CI; 1.76, 46.1) to be satisfied for antenatal services than those who were in the first visit. Women with family monthly income of $US 25-100 per month were 60% (AOR = 0.4, 95% CI; 0.2, 0.8) less likely to have satisfaction by skilled antenatal care services than those who had monthly household income below $US 25., Conclusion and Recommendation: Women who reported good-satisfaction to overall skilled antenatal care services were highest as compared to previous Ethiopian study findings. Demographic, economic, obstetric and distance factors were independent predictors of satisfaction to skilled antenatal care services. Non natives must be encouraged to seek satisfying services.

Objective: Limb-girdle muscular dystrophies (LGMDs), one of the most heterogeneous neuromuscular disorders (NMDs), involves predominantly proximal-muscle weakness with >30 genes associated with different subtypes. The clinical-genetic overlap among subtypes and with other NMDs complicate disease-subtype identification lengthening diagnostic process, increases overall costs hindering treatment/clinical-trial recruitment. Currently seven LGMD clinical trials are active but still no gene-therapy-related treatment is available. Till-date no nation-wide large-scale LGMD sequencing program was performed. Our objectives were to understand LGMD genetic basis, different subtypes' relative prevalence across US and investigate underlying disease mechanisms., Methods: A total of 4656 patients with clinically suspected-LGMD across US were recruited to conduct next-generation sequencing (NGS)-based gene-panel testing during June-2015 to June-2017 in CLIA-CAP-certified Emory-Genetics-Laboratory. Thirty-five LGMD-subtypes-associated or LGMD-like other NMD-associated genes were investigated. Main outcomes were diagnostic yield, gene-variant spectrum, and LGMD subtypes' prevalence in a large US LGMD-suspected population., Results: Molecular diagnosis was established in 27% (1259 cases; 95% CI, 26-29%) of the patients with major contributing genes to LGMD phenotypes being: CAPN3 (17%), DYSF (16%), FKRP (9%) and ANO5 (7%). We observed an increased prevalence of genetically confirmed late-onset Pompe disease, DNAJB6- associated LGMD subtype1E and CAPN3 -associated autosomal-dominant LGMDs. Interestingly, we identified a high prevalence of patients with pathogenic variants in more than one LGMD gene suggesting possible synergistic heterozygosity/digenic/multigenic contribution to disease presentation/progression that needs consideration as a part of diagnostic modality., Interpretation: Overall, this study has improved our understanding of the relative prevalence of different LGMD subtypes, their respective genetic etiology, and the changing paradigm of their inheritance modes and novel mechanisms that will allow for improved timely treatment, management, and enrolment of molecularly diagnosed individuals in clinical trials.

Wagner syndrome and erosive vitreoretinopathy together constitute the phenotypic continuum of an autosomal dominant vitreoretinopathy, with clinical findings typically isolated to the eye. The disease is caused by pathogenic variants in the VCAN gene and all such variants reported to date are those that plausibly result in haploinsufficiency of exon 8 containing vcan transcripts. Here, we report the molecular findings and long-term follow-up of a 16-year-old female with a history of retinal detachments and pigmentary retinal changes. Next-generation sequencing and microarray analysis of 141 genes established a diagnosis of Wagner syndrome in this individual, by detection of an 11.7 kilobase (kb) deletion encompassing exon 8 of VCAN. In light of the emerging functions and roles of versican protein in human disease, we discuss how variants within exon 8 of the VCAN gene can be compared to those in exon 2 of the COL2A1 gene that cause atypical Stickler syndrome and propose that variants in other regions of the gene can be expected to present with a more systemic disease. The distinctive facial features and atypical gastrointestinal symptoms observed in this long-term follow-up study support the possibility that individuals with VCAN-related vitreoretinopathy may have extra-ocular clinical features., (© 2018 Wiley Periodicals, Inc.)

GM2 activator (GM2A) deficiency (OMIM 613109) is a rare lysosomal storage disorder, with onset typically in infancy or early childhood. Clinically, it is almost indistinguishable from Tay-Sachs disease (OMIM 272800) or Sandhoff disease (OMIM 268800); however, traditionally available biochemical screening tests will most likely reveal normal results. We report a 2-year-old male with initially normal development until the age of 9 months, when he presented with developmental delay and regression. Workup at that time was unrevealing; at 15 months, he had abnormal brain MRI findings and a cherry red spot on ophthalmological examination. Family history and all laboratory studies were uninformative. The combination of a cherry red spot and developmental regression was strongly suggestive of a lysosomal storage disorder. Sequence analysis of GM2A did not reveal any pathogenic variants; however, exon 2 of GM2A could not be amplified by PCR, raising suspicion for a large, homozygous deletion. Subsequent copy number analysis confirmed a homozygous deletion of exon 2 in GM2A. This is the first reported case of GM2A deficiency being caused by a whole exon deletion. We describe previously unreported electron microscopy findings in this disease, thus expanding the clinical and variant spectrum for GM2 activator deficiency. These findings demonstrate the increased degree of suspicion required for diagnosis of this rare disorder. Brief Summary: This case of GM2 activator deficiency was caused by a homozygous deletion in GM2A, demonstrating the need to include exon level copy number analysis in any workup to fully exclude this disorder.

Background: Limb-girdle muscular dystrophy (LGMD) is the most common adult-onset class of muscular dystrophies in India, but a majority of suspected LGMDs in India remain unclassified to the genetic subtype level. The next-generation sequencing (NGS)-based approaches have allowed molecular characterization and subtype diagnosis in a majority of these patients in India., Materials and Methods: (I) To select probable dysferlinopathy (LGMD2B) cases from other LGMD subtypes using two screening methods (i) to determine the status of dysferlin protein expression in blood (peripheral blood mononuclear cell) by monocyte assay (ii) using a predictive algorithm called automated LGMD diagnostic assistant (ALDA) to obtain possible LGMD subtypes based on clinical symptoms. (II) Identification of gene pathogenic variants by NGS for 34 genes associated with LGMD or LGMD like muscular dystrophies, in cases showing: absence of dysferlin protein by the monocyte assay and/or a typical dysferlinopathy phenotype, with medium to high predictive scores using the ALDA tool., Results: Out of the 125 patients screened by NGS, 96 were confirmed with two dysferlin variants, of which 84 were homozygous. Single dysferlin pathogenic variants were seen in 4 patients, whereas 25 showed no variants in the dysferlin gene., Conclusion: In this study, 98.2% of patients with absence of the dysferlin protein showed one or more variants in the dysferlin gene and hence has a high predictive significance in diagnosing dysferlinopathies. However, collection of blood samples from all over India for protein analysis is expensive. Our analysis shows that the use of the "ALDA tool" could be a cost-effective alternative method. Identification of dysferlin pathogenic variants by NGS is the ultimate method for diagnosing dysferlinopathies though follow-up with the monocyte assay can be useful to understand the phenotype in relation to the dysferlin protein expression and also be a useful biomarker for future clinical trials., Competing Interests: There are no conflicts of interest.

Purpose: Next-generation sequencing (NGS) is now routinely used to interrogate large sets of genes in a diagnostic setting. Regions of high sequence homology continue to be a major challenge for short-read technologies and can lead to false-positive and false-negative diagnostic errors. At the scale of whole-exome sequencing (WES), laboratories may be limited in their knowledge of genes and regions that pose technical hurdles due to high homology. We have created an exome-wide resource that catalogs highly homologous regions that is tailored toward diagnostic applications., Methods: This resource was developed using a mappability-based approach tailored to current Sanger and NGS protocols., Results: Gene-level and exon-level lists delineate regions that are difficult or impossible to analyze via standard NGS. These regions are ranked by degree of affectedness, annotated for medical relevance, and classified by the type of homology (within-gene, different functional gene, known pseudogene, uncharacterized noncoding region). Additionally, we provide a list of exons that cannot be analyzed by short-amplicon Sanger sequencing., Conclusion: This resource can help guide clinical test design, supplemental assay implementation, and results interpretation in the context of high homology.Genet Med 18 12, 1282-1289.

Next-generation sequencing is changing the paradigm of clinical genetic testing. Today there are numerous molecular tests available, including single-gene tests, gene panels, and exome sequencing or genome sequencing. As a result, ordering physicians face the conundrum of selecting the best diagnostic tool for their patients with genetic conditions. Single-gene testing is often most appropriate for conditions with distinctive clinical features and minimal locus heterogeneity. Next-generation sequencing-based gene panel testing, which can be complemented with array comparative genomic hybridization and other ancillary methods, provides a comprehensive and feasible approach for heterogeneous disorders. Exome sequencing and genome sequencing have the advantage of being unbiased regarding what set of genes is analyzed, enabling parallel interrogation of most of the genes in the human genome. However, current limitations of next-generation sequencing technology and our variant interpretation capabilities caution us against offering exome sequencing or genome sequencing as either stand-alone or first-choice diagnostic approaches. A growing interest in personalized medicine calls for the application of genome sequencing in clinical diagnostics, but major challenges must be addressed before its full potential can be realized. Here, we propose a testing algorithm to help clinicians opt for the most appropriate molecular diagnostic tool for each scenario.

The field of clinical genetics has advanced at an unprecedented pace. Today, with the aid of several high-resolution and high-precision technologies, physicians are able to make molecular genetic diagnoses for many infants affected with genetic disease. It is imperative, however, that perinatologists and neonatologists understand the strengths and limitations of genetic testing. This article discusses the different genetic testing options available for perinatal and neonatal diagnostics, along with their clinical utilities and indications. From variant-specific testing to whole-exome and genome sequencing, the article covers the whole gamut of genetic testing, with some thoughts on the changing paradigm of medical genetics., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Nemaline myopathy (NM) is a congenital neuromuscular disorder often characterized by hypotonia, facial weakness, skeletal muscle weakness, and the presence of rods on muscle biopsy. A rare form of nemaline myopathy known as Amish Nemaline Myopathy has only been seen in a genetically isolated cohort of Old Order Amish patients who may additionally present with tremors in the first 2-3 months of life., Methods: We describe an Hispanic male diagnosed with nemaline myopathy histopathologically and subsequently confirmed by next generation gene sequencing., Results: Direct sequencing revealed that he is homozygous for a pathogenic nonsense variant c.323C>G (p.S108X) in exon 9 of the TNNT1 gene., Conclusions: This report describes a novel pathogenic variant in the TNNT1 gene and represents a nemaline myopathy-causing variant in the TNNT1 gene outside of the Old Order Amish and Dutch ancestry., (© 2014 Wiley Periodicals, Inc.)

Objective: Neuromuscular diseases (NMDs) are a group of >200 highly genetically as well as clinically heterogeneous inherited genetic disorders that affect the peripheral nervous and muscular systems, resulting in gross motor disability. The clinical and genetic heterogeneities of NMDs make disease diagnosis complicated and expensive, often involving multiple tests., Methods: To expedite the molecular diagnosis of NMDs, we designed and validated several next generation sequencing (NGS)-based comprehensive gene panel tests that include complementary deletion and duplication testing through comparative genomic hybridization arrays. Our validation established the targeted gene panel test to have 100% sensitivity and specificity for single nucleotide variant detection. To compare the clinical diagnostic yields of single gene (NMD-associated) tests with the various NMD NGS panel tests, we analyzed data from all clinical tests performed at the Emory Genetics Laboratory from October 2009 through May 2014. We further compared the clinical utility of the targeted NGS panel test with that of exome sequencing (ES)., Results: We found that NMD comprehensive panel testing has a 3-fold greater diagnostic yield (46%) than single gene testing (15-19%). Sanger fill-in of low-coverage exons, copy number variation analysis, and thorough in-house validation of the assay all complement panel testing and allow the detection of all types of causative pathogenic variants, some of which (about 18%) may be missed by ES., Interpretation: Our results strongly indicate that for molecular diagnosis of heterogeneous disorders such as NMDs, targeted panel testing has the highest clinical yield and should therefore be the preferred first-tier approach., (© 2014 American Neurological Association.)

Background: Dihydropyrimidine dehydrogenase (DPD) encoded by DPYD gene is the major enzyme involved in metabolism of 5-flurouracil (5-FU), a pyrimidine analogue used in cancer chemotherapy. Although very effective as a cancer therapeutic drug, if not rapidly metabolized, 5-FU may prove lethal. Single nucleotide variants (SNVs) within DPYD that modulate DPD enzyme activity contribute to 5-FU toxicity., Study: This study looked for DPYD SNVs common in the Indian population that might be associated with variable DPD activity and drug toxicity. To achieve this, sequencing analysis was performed of all 23 exons and flanking intronic regions of the DPYD gene in a cohort of 50 healthy adult Indians. This study detected 22 SNVs including intronic, synonymous and non-synonymous changes in the DPYD gene, of which six have not been documented before. Allelic frequency was calculated for the observed variants and linkage disequilibrium (LD) analysis was performed on variants with frequency ≥0.1 to identify haplotypes., Conclusions: This study provides a brief overview of the genetic polymorphism in DPYD in Indians and emphasizes the need for a large scale extensive study to establish markers associated with the frequently observed variable drug metabolism.

South Asian Indians represent a sixth of the world's population and are a racially, geographically, and genetically diverse people. Their unique anthropological structure, prevailing caste system, and ancient religious practices have all impacted the genetic composition of most of the current-day Indian population. With the evolving socio-religious and economic activities of the subsects and castes, endogamous and consanguineous marriages became a commonplace. Consequently, the frequency of founder mutations and the burden of heritable genetic disorders rose significantly. Specifically, the incidence of certain autosomal-recessive disorders is relatively high in select Indian subpopulations and communities that share common recent ancestry. Although today clinical genetics and molecular diagnostic services are making inroads in India, the high costs associated with the technology and the tests often keep patients from an exact molecular diagnosis, making more customized and tailored tests, such as those interrogating the most common and founder mutations or those that cater to select sects within the population, highly attractive. These tests offer a quick first-hand affordable diagnostic and carrier screening tool. Here, we provide a comprehensive catalog of known common mutations and founder mutations in the Indian population and discuss them from a molecular, clinical, and historical perspective., (© 2014 WILEY PERIODICALS, INC.)

Duchenne Muscular Dystrophy (DMD) is an X-linked inherited neuromuscular disorder caused by mutations in the dystrophin gene (DMD; locus Xp21.2). The mutation spectrum of DMD is unique in that 65% of causative mutations are intragenic deletions, with intragenic duplications and point mutations (along with other sequence variants) accounting for 6% to 10% and 30% to 35%, respectively. The strategy for molecular diagnostic testing for DMD involves initial screening for deletions/duplications using microarray-based comparative genomic hybridization (array-CGH) followed by full-sequence analysis of DMD for sequence variants. Recently, next-generation sequencing (NGS)-based targeted gene analysis has become clinically available for detection of point mutations and other sequence variants (small insertions, deletions, and indels). This unit initially discusses the strategic algorithm for establishing a molecular diagnosis of DMD and later provides detailed protocols of current molecular diagnostic methods for DMD, including array-CGH, PCR-based Sanger sequencing, and NGS-based sequencing assay., (Copyright © 2014 John Wiley & Sons, Inc.)

Introduction: Dysferlin deficiency causes dysferlinopathies. Among peripheral blood mononuclear cells (PBMCs), the dysferlin protein is expressed specifically in CD14(+) monocytes., Methods: We quantified dysferlin protein levels in PBMC lysates of 77 individuals suspected clinically of having a dysferlinopathy to screen for true positives. Subsequent molecular confirmation was done by Sanger sequencing and comparative genomic hybridization arrays to establish diagnosis., Results: Of the 44 individuals who had significantly reduced dysferlin levels (≤10%), 41 underwent molecular testing. We identified at least 1 mutation in 85% (35 of 41), and 2 mutations, establishing a dysferlinopathy diagnosis, in 61% (25 of 41) of these individuals. Among those with dysferlin protein levels of >10% (33 of 77), only 1 individual (of 14 who underwent molecular testing) had a detectable mutation., Conclusions: Our results suggest that dysferlin protein levels of ≤10% in PBMCs, are highly indicative of primary dysferlinopathies. However, this assay may not distinguish carriers from those with secondary dysferlin reduction., (© 2014 Wiley Periodicals, Inc.)

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle, and peripheral blood monocytes. Expression in skeletal muscle and monocytes strongly correlates in healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While 18 of these individuals with protein levels in the range of 40%-64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of 18 detected missense variants in only four. Analysis of DNA methylation patterns at the DYSF locus showed no changes in methylation levels at CpG islands and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, the monocyte assay cannot be used to determine the carrier status; for this, a molecular analysis of DYSF must be performed., (© 2014 WILEY PERIODICALS, INC.)

Cytidine deaminase (CDA) is the major enzyme involved in metabolism of gemcitabine, a pyrimidine analog widely used for chemotherapy of solid tumors. While only low amounts of administered gemcitabine undergo intracellular phosphorylation into active forms and involve in antineoplastic activities, majority of it is rapidly inactivated by CDA and excreted to avoid drug toxicity. Knowledge of the genetic polymorphisms mildly effecting cellular activity of the enzyme CDA is therefore crucial to understanding drug-induced toxicities associated with gemcitabine. Functional significance and allele frequencies for common SNPs including 79A>C (*2) and 208G>A (*3) have been reported in various ethnic populations including Caucasian, African, Korean and Japanese. However, such studies have not been reported in any Indian sub-population. In the present study, conventional polymerase chain reaction (PCR) based amplification using gene specific primers and Sanger sequencing were performed to identify CDA variants in 50 healthy individuals from Indian sub-population. Established common variant 79A>C known to reduce CDA activity was observed at a frequency of 0.14 in the study cohort. In addition to other known variants, one novel variant, c.325-209T>C was detected at a frequency of 0.06. Genetic variants in CDA gene and their frequencies established in our study hold value in pharmacogenetics., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Introduction: Clinical heterogeneity of limb-girdle muscular dystrophies (LGMDs, 24 known subtypes), which includes overlapping phenotypes and varying ages of onset and morbidity, adds complexity to clinical and molecular diagnoses., Methods: To diagnose LGMD subtype, protein analysis, using immunohistochemistry (IHC) and immunoblotting, was followed by gene sequencing through a panel approach (simultaneous sequencing of known LGMD genes) in 9 patients from unrelated families of the Indian Agarwal community. Haplotype studies were performed by targeted SNP genotyping to establish mutation segregation., Results: We identified 2 founder mutations in CAPN3, a missense (c.2338G>C; p.D780H) and a splice-site (c.2099-1G>T) mutation, on 2 different haplotype backgrounds. The patients were either heterozygous for both or homozygous for either of these mutations., Conclusions: Founder mutations have immediate clinical application, at least in selected population groups. Clinically available gene panels may provide a definitive molecular diagnosis for heterogeneous disorders such as LGMD., (Copyright © 2012 Wiley Periodicals, Inc.)

Many studies have documented the induction of belowground defenses in plants in response to aboveground herbivory and vice versa, but the genes and signaling molecules mediating systemic induction are not well understood. We performed comparative microarray analysis on maize whorl and root tissues from the insect resistant inbred Mp708 in response to foliar feeding by fall armyworm (Spodoptera frugiperda) caterpillars. Although Mp708 has elevated jasmonic acid (JA) levels prior to herbivory, genes involved in JA biosynthesis were up-regulated in whorls in response to fall armyworm feeding. Alternatively, genes possibly involved in regulating ethylene (ET) perception and signaling were up-regulated in roots following foliar herbivory. Transcript levels of genes encoding proteins involved in direct defenses against herbivores were enhanced both in roots and leaves, but transcriptional factors and genes involved in various biosynthetic pathways were selectively down-regulated in the whorl. The results indicate that foliar herbivory by fall armyworm changes root gene expression pathways suggesting profound long distance signaling. Tissue specific induction and suppression of JA and ET signaling pathway genes provides a clue to their possible roles in signaling between the two distant tissue types that eventually triggers defense responses in the roots in response to foliar herbivory., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications. Using a highly multiplexed PCR-based target enrichment method (RainDance) in conjunction with NGS, we performed mutation detection in all CMD genes of 26 samples and compared the results with Sanger sequencing. The RainDance NGS panel showed great consistency in coverage depth, on-target efficiency, versatility of mutation detection, and genotype concordance with Sanger sequencing, demonstrating the test's appropriateness for clinical use. Compared to single tests, a higher diagnostic yield was observed by panel implementation. The panel's limitation is the amplification failure of select gene-specific exons which require Sanger sequencing for test completion. Successful validation and application of the CMD NGS panel to improve the diagnostic yield in a clinical laboratory was shown.

Although biallelic mutations in non-collagen genes account for <10% of individuals with osteogenesis imperfecta, the characterization of these genes has identified new pathways and potential interventions that could benefit even those with mutations in type I collagen genes. We identified mutations in FKBP10, which encodes the 65 kDa prolyl cis-trans isomerase, FKBP65, in 38 members of 21 families with OI. These include 10 families from the Samoan Islands who share a founder mutation. Of the mutations, three are missense; the remainder either introduce premature termination codons or create frameshifts both of which result in mRNA instability. In four families missense mutations result in loss of most of the protein. The clinical effects of these mutations are short stature, a high incidence of joint contractures at birth and progressive scoliosis and fractures, but there is remarkable variability in phenotype even within families. The loss of the activity of FKBP65 has several effects: type I procollagen secretion is slightly delayed, the stabilization of the intact trimer is incomplete and there is diminished hydroxylation of the telopeptide lysyl residues involved in intermolecular cross-link formation in bone. The phenotype overlaps with that seen with mutations in PLOD2 (Bruck syndrome II), which encodes LH2, the enzyme that hydroxylates the telopeptide lysyl residues. These findings define a set of genes, FKBP10, PLOD2 and SERPINH1, that act during procollagen maturation to contribute to molecular stability and post-translational modification of type I procollagen, without which bone mass and quality are abnormal and fractures and contractures result.

Background: Aspergillus flavus infection and aflatoxin contamination of maize pose negative impacts in agriculture and health. Commercial maize hybrids are generally susceptible to this fungus. Significant levels of host plant resistance have been observed in certain maize inbred lines. This study was conducted to identify maize genes associated with host plant resistance or susceptibility to A. flavus infection and aflatoxin accumulation., Results: Genome wide gene expression levels with or without A. flavus inoculation were compared in two resistant maize inbred lines (Mp313E and Mp04:86) in contrast to two susceptible maize inbred lines (Va35 and B73) by microarray analysis. Principal component analysis (PCA) was used to find genes contributing to the larger variances associated with the resistant or susceptible maize inbred lines. The significance levels of gene expression were determined by using SAS and LIMMA programs. Fifty candidate genes were selected and further investigated by quantitative RT-PCR (qRT-PCR) in a time-course study on Mp313E and Va35. Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E., Conclusion: Maize genes associated with host plant resistance or susceptibility were identified by a combination of microarray analysis, qRT-PCR analysis, and QTL mapping methods. Our findings suggest that multiple mechanisms are involved in maize host plant defense systems in response to Aspergillus flavus infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation.

Non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), and microhomology-mediated replication-dependent recombination (MMRDR) have all been put forward as mechanisms to explain DNA rearrangements associated with genomic disorders. However, many nonrecurrent rearrangements in humans remain unexplained. To further investigate the mutation mechanisms of these copy number variations (CNVs), we performed breakpoint mapping analysis for 62 clinical cases with intragenic deletions in the human DMD gene (50 cases) and other known disease-causing genes (one PCCB, one IVD, one DBT, three PAH, one STK11, one HEXB, three DBT, one HRPT1, and one EMD cases). While repetitive elements were found in only four individual cases, three involving DMD and one HEXB gene, microhomologies (2-10 bp) were observed at breakpoint junctions in 56% and insertions ranging from 1 to 48 bp were seen in 16 of the total 62 cases. Among these insertions, we observed evidence for tandem repetitions of short segments (5-20 bp) of reference sequence proximal to the breakpoints in six individual DMD cases (six repeats in one, four repeats in three, two repeats in one, and one repeat in one case), strongly indicating attempts by the replication machinery to surpass the stalled replication fork. We provide evidence of a novel template slippage event during replication rescue. With a deeper insight into the complex process of replication and its rescue during origin failure, brought forward by recent studies, we propose a hypothesis based on aberrant firing of replication origins to explain intragenic nonrecurrent rearrangements within genes, including the DMD gene.

Insect pests that attempt to feed on the caterpillar-resistant maize genotype Mp708 encounter a potent, multipronged defense system that thwarts their invasion. First, these plants are on "constant alert" due to constitutively elevated levels of the phytohormone jasmonic acid that signals the plant to activate its defenses. The higher jasmonic acid levels trigger the expression of defense genes prior to herbivore attack so the plants are "primed" and respond with a faster and stronger defense. The second defense is the rapid accumulation of a toxic cysteine protease called Mir1-CP in the maize whorl in response to caterpillar feeding. When caterpillars ingest Mir1-CP, it damages the insect's midgut and retards their growth. In this article, we discuss a third possible defense strategy employed by Mp708. We have shown that foliar caterpillar feeding causes Mir1-CP and defense gene transcripts to accumulate in its roots. We propose that caterpillar feeding aboveground sends a signal belowground via the phloem that results in Mir1-CP accumulation in the roots. We also postulate that the roots serve as a reservoir of Mir1-CP that can be mobilized to the whorl in response to caterpillar assault.

Background: Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin., Results: In order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus., Conclusions: CFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query options for mining the database across different types of experiments. The database is publically available at http://agbase.msstate.edu.

This study was conducted to determine if constitutive levels of jasmonic acid (JA) and other octadecanoid compounds were elevated prior to herbivory in a maize genotype with documented resistance to fall armyworm (Spodoptera frugiperda) and other lepidopteran pests. The resistant inbred Mp708 had approximately 3-fold higher levels of jasmonic acid (JA) prior to herbivore feeding than the susceptible inbred Tx601. Constitutive levels of cis-12-oxo-phytodienoic acid (OPDA) also were higher in Mp708 than Tx601. In addition, the constitutive expression of JA-inducible genes, including those in the JA biosynthetic pathway, was higher in Mp708 than Tx601. In response to herbivory, Mp708 generated comparatively higher levels of hydrogen peroxide, and had a greater abundance of NADPH oxidase transcripts before and after caterpillar feeding. Before herbivore feeding, low levels of transcripts encoding the maize insect resistance cysteine protease (Mir1-CP) and the Mir1-CP protein were detected consistently. Thus, Mp708 appears to have a portion of its defense pathway primed, which results in constitutive defenses and the ability to mount a stronger defense when caterpillars attack. Although the molecular mechanisms that regulate the constitutive accumulation of JA in Mp708 are unknown, it might account for its enhanced resistance to lepidopteran pests. This genotype could be valuable in studying the signaling pathways that maize uses to response to insect herbivores.

In plants, ethylene and jasmonate control the defense responses to multiple stressors, including insect predation. Among the defense proteins known to be regulated by ethylene is maize insect resistance 1-cysteine protease (Mir1-CP). This protein is constitutively expressed in the insect-resistant maize (Zea mays) genotype Mp708; however, its abundance significantly increases during fall armyworm (Spodoptera frugiperda) herbivory. Within 1 h of herbivory by fall armyworm, Mir1-CP accumulates at the feeding site and continues to increase in abundance until 24 h without any increase in its transcript (mir1) levels. To resolve this discrepancy and elucidate the role of ethylene and jasmonate in the signaling of Mir1-CP expression, the effects of phytohormone biosynthesis and perception inhibitors on Mir1-CP expression were tested. Immunoblot analysis of Mir1-CP accumulation and quantitative reverse-transcriptase polymerase chain reaction examination of mir1 levels in these treated plants demonstrate that Mir1-CP accumulation is regulated by both transcript abundance and protein expression levels. The results also suggest that jasmonate functions upstream of ethylene in the Mir1-CP expression pathway, allowing for both low-level constitutive expression and a two-stage defensive response, an immediate response involving Mir1-CP accumulation and a delayed response inducing mir1 transcript expression.

When lepidopteran larvae feed on the insect-resistant maize genotype Mp708 there is a rapid accumulation of a defensive cysteine protease, Maize insect resistance 1-cysteine protease (Mir1-CP), at the feeding site. Silver-enhanced immunolocalization visualized with both light and transmission electron microscopy was used to determine the location of Mir1-CP in the maize leaf. The results indicated that Mir1-CP is localized predominantly in the phloem of minor and intermediate veins. After 24 h of larval feeding, Mir1-CP increased in abundance in the vascular parenchyma cells and in the thick-walled sieve element (TSE); it was also found localized to the bundle sheath and mesophyll cells. In situ hybridization of mRNA encoding Mir1-CP indicated that the primary sites of Mir1-CP synthesis in the whorl are the vascular parenchyma and bundle sheath cells. In addition to the phloem, Mir1-CP was also found in the metaxylem of the leaf and root. After 24 h of foliar feeding, the amount of Mir1-CP in the root xylem increased and it appeared to move from xylem parenchyma into the root metaxylem elements. The accumulation of Mir1-CP in maize vascular elements suggests Mir1-CP may move through these tissues to defend against insect herbivores.