Resolving misalignment interference for NGS-based clinical diagnostics.

Next-generation sequencing (NGS) is an incredibly useful tool for genetic disease diagnosis. However, the most commonly used bioinformatics methods for analyzing sequence reads insufficiently discriminate genomic regions with extensive sequence identity, such as gene families and pseudogenes, complicating diagnostics. This problem has been recognized for specific genes, including many involved in human disease, and diagnostic labs must perform additional costly steps to guarantee accurate diagnosis in these cases. Here we report a new data analysis method based on the comparison of read depth between highly homologous regions to identify misalignment. Analyzing six clinically important genes-CYP21A2, GBA, HBA1/2, PMS2, and SMN1-each exhibiting misalignment issues related to homology, we show that our technique can correctly identify potential misalignment events and be used to make appropriate calls. Combined with long-range PCR and/or MLPA orthogonal testing, our clinical laboratory can improve variant calling with minimal additional cost. We propose an accurate and cost-efficient NGS testing procedure that will benefit disease diagnostics, carrier screening, and research-based population studies.

Alterations in Brca1 expression in mouse ovarian granulosa cells have short-term and long-term consequences on estrogen-responsive organs.

Incessant menstrual cycle activity, uninterrupted by either pregnancy or oral contraceptive use, is the most important risk factor for sporadic ovarian cancer. Menstrual cycle progression is partly controlled by steroid hormones such as estrogens and others that are secreted by the ovarian granulosa cells. We showed earlier that mice carrying a homozygous granulosa cell-specific knockout of Brca1, the homolog of BRCA1 that is associated with familial ovarian cancer predisposition in humans, develop benign epithelial tumors in their reproductive tract. These tumors are driven, at least in part, by a prolongation of the proestrus phase of the estrus cycle (equivalent to the follicular phase of the menstrual cycle) in Brca1 mutant mice, resulting in prolonged unopposed estrogen stimulation. Mutant mice synchronized in proestrus also showed increased circulating estradiol levels, but the possibility that this change also has a role in tumor predisposition was not investigated. We sought to determine whether these changes in hormonal stimulation result in measurable changes in tissues targeted by estrogen outside the ovary. Here we show that mice carrying a Brca1 mutation in their ovarian granulosa cells show increased endometrial proliferation during proestrus, implying that the effects of Brca1 inactivation on estrogen stimulation have short-term consequences, at least on this target organ. We further show that mutant mice develop increased femoral trabecular thickness and femoral length, which are well-known consequences of chronic estrogen stimulation. Estrogen biosynthesis by granulosa cells was increased not only in mice carrying a homozygous Brca1 mutation, but also in heterozygous mutants mimicking the mutational status in granulosa cells of human BRCA1 mutation carriers. The results suggest that human germline BRCA1 mutations, although associated with increased cancer risk, may also have beneficial consequences, such as increased bone strength, that may have contributed to the maintenance of mutated BRCA1 alleles in the human gene pool.

Genetic abnormalities in biopsy-proven, adult-onset hemolytic uremic syndrome and C3 glomerulopathy.

Atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) have been linked to mutations in many of the proteins that are involved in alternative complement pathway activation. Age and etiology confounded, the prevalence of such mutations has been reported to be over 30 to 50% in these diseases. However, the cohorts studied included many children or individuals with a familial history of complement-related disorders and genetic tests were usually limited to exome sequencing of known causative or risk-associated genes. In this study, a retrospective adult cohort of 35 patients with biopsy-proven thrombotic microangiopathy (the largest in Canada) and 10 patients with C3 glomerulopathy was tested through an extended exome panel to identify causative defects in associated or candidate genes including those of the alternative and terminal complement pathways. A variant of unknown significance was also analyzed for pathogenicity through in vitro studies. To our surprise, the prevalence of known causative or risk-associated variants in either of these cohorts was found to be less than ~ 15% overall. However, the panel used and analyses carried out allowed to identify novel variants of potential clinical significance and a number of candidate genes. The prevalence of known genetic defects in adult-onset aHUS and C3G is thus probably much lower than 30 to 50%. Our results also point towards the importance of investigating diseases of the alternative complement pathway through extended exome panels and in vitro analyses. KEY MESSAGES: The alternative complement pathway plays a major role in the pathogenesis of hemolytic uremic syndrome and C3 glomerulopathy. Based on previous studies, both disorders have been commonly linked to variants in the various intermediates that sustain or regulate this pathway. The prevalence of such mutations in the adult-onset and sporadic forms of these diseases is probably much lower than expected based on larger series. The sporadic forms of complementopathies are likely to involve additional genes that are yet to be uncovered.

Jagged1 functions downstream of Twist1 in the specification of the coronal suture and the formation of a boundary between osteogenic and non-osteogenic cells.

The Notch pathway is crucial for a wide variety of developmental processes including the formation of tissue boundaries. That it may function in calvarial suture development and figure in the pathophysiology of craniosynostosis was suggested by the demonstration that heterozygous loss of function of JAGGED1 in humans can cause Alagille syndrome, which has craniosynostosis as a feature. We used conditional gene targeting to examine the role of Jagged1 in the development of the skull vault. We demonstrate that Jagged1 is expressed in a layer of mesoderm-derived sutural cells that lie along the osteogenic-non-osteogenic boundary. We show that inactivation of Jagged1 in the mesodermal compartment of the coronal suture, but not in the neural crest compartment, results in craniosynostosis. Mesodermal inactivation of Jagged1 also results in changes in the identity of sutural cells prior to overt osteogenic differentiation, as well as defects in the boundary between osteogenic and non-osteogenic compartments at the coronal suture. These changes, surprisingly, are associated with increased expression of Notch2 and the Notch effector, Hes1, in the sutural mesenchyme. They are also associated with an increase in nuclear ß-catenin. In Twist1 mutants, Jagged1 expression in the suture is reduced substantially, suggesting an epistatic relationship between Twist1 and Jagged1. Consistent with such a relationship, Twist1-Jagged1 double heterozygotes exhibit a substantial increase in the severity of craniosynostosis over individual heterozygotes. Our results thus suggest that Jagged1 is an effector of Twist1 in coronal suture development.

A Streamlined Approach to Prader-Willi and Angelman Syndrome Molecular Diagnostics.

Establishing or ruling out a molecular diagnosis of Prader-Willi or Angelman syndrome (PWS/AS) presents unique challenges due to the variety of different genetic alterations that can lead to these conditions. Point mutations, copy number changes, uniparental isodisomy (i-UPD) 15 of two subclasses (segmental or total isodisomy), uniparental heterodisomy (h-UPD), and defects in the chromosome 15 imprinting center can all cause PWS/AS. Here, we outline a combined approach using whole-exome sequencing (WES) and DNA methylation data with methylation-sensitive multiplex ligation-dependent probe amplification (MLPA) to establish both the disease diagnosis and the mechanism of disease with high sensitivity using current standard of care technology and improved efficiency compared to serial methods. The authors encourage the use of this approach in the clinical setting to confirm and establish the diagnosis and genetic defect which may account for the secondary genetic conditions that may be seen in those with isodisomy 15, impacting surveillance and counseling with more accurate recurrence risks. Other similarly affected individuals due to other gene disorders or cytogenetic anomalies such as Rett syndrome or microdeletions would also be identified with this streamlined approach.

The Value of Parental Testing by Next-Generation Sequencing Includes the Detection of Germline Mosaicism.

When a potential disease-causing variant is detected in a proband, parental testing is used to determine the mode of inheritance. This study demonstrates that next-generation sequencing (NGS) is uniquely well suited for parental testing, in particular because of its ability to detect clinically relevant germline mosaicism. Parental variant testing by NGS was performed in a clinical laboratory for 1 year. The detection of mosaicism by NGS was compared with its detection by Sanger sequencing. Eight cases of previously unrevealed mosaicism were detected by NGS across eight different genes. Mosaic variants were differentiated from sequencing noise using custom bioinformatics analyses in combination with familial inheritance data and complementary Sanger sequencing. Sanger sequencing detected mosaic variants with allele fractions ≥8% by NGS, but could not detect mosaic variants below that level. Detection of germline mosaicism by NGS is invaluable to parents, providing a more accurate recurrence risk that can alter decisions on family planning and pregnancy management. Because NGS can also confirm parentage and increase scalability, it simultaneously streamlines and strengthens the variant curation process. These features make NGS the ideal method for parental testing, superior even to Sanger sequencing for most genomic loci.

Supraventricular tachycardias, conduction disease, and cardiomyopathy in 3 families with the same rare variant in TNNI3K (p.Glu768Lys).

BACKGROUND: Rare genetic variants in TNNI3K encoding troponin-I interacting kinase have been linked to a distinct syndrome consisting primarily of supraventricular tachycardias and variably expressed conduction disturbance and dilated cardiomyopathy in 2 families. OBJECTIVE: The purpose of this study was to identify new genetic variants associated with inherited supraventricular tachycardias, cardiac conduction disease, and cardiomyopathy. METHODS: We conducted next generation sequencing in 3 independent multigenerational families with atrial/junctional tachycardia with or without conduction disturbance, dilated cardiomyopathy, and sudden death. We also assessed the effect of identified variant on protein autophosphorylation. RESULTS: In this study, we uncovered the same ultra-rare genetic variant in TNNI3K (c.2302G>A, p.Glu768Lys), which co-segregated with disease features in all affected individuals (n = 23) from all 3 families. TNNI3K harboring the TNNI3K-p.Glu768Lys variant displayed enhanced kinase activity, in line with expectations from previous mouse studies that demonstrated increased conduction indices and procardiomyopathic effects with increased levels of Tnni3k. CONCLUSION: This study corroborates further the causal link between rare genetic variation in TNNI3K and this distinct complex phenotype, and points to enhanced kinase activity of TNNI3K as the underlying pathobiological mechanism.

Cell-nonautonomous induction of ovarian and uterine serous cystadenomas in mice lacking a functional Brca1 in ovarian granulosa cells.

Women with germline mutations in BRCA1 have a 40% risk of developing ovarian cancer by age 70 and are also predisposed to cancers of the fallopian tubes. Given that ovulatory activity is a strong risk factor for sporadic ovarian cancer, we hypothesized that reduced BRCA1 expression might predispose to gynecological cancers indirectly, by influencing ovarian granulosa cells. These cells secrete sex steroids that control the ovulatory cycle and influence the growth of ovarian epithelial tumors. Granulosa cells also secrete mullerian inhibiting substance (MIS), a hormone that inhibits both the formation of female reproductive organs in male embryos and the proliferation of ovarian epithelial tumor cells. We tested this hypothesis by using the Cre-lox system to inactivate the Brca1 gene in mouse ovarian granulosa cells. A truncated form of the Fsh receptor promoter served as the Cre driver. Here, we show that indeed, inactivation of the Brca1 gene in granulosa cells led to the development of cystic tumors in the ovaries and uterine horns. These tumors carried normal Brca1 alleles, supporting the view that Brca1 may influence tumor development indirectly, possibly through an effector secreted by granulosa cells.

Requirement for Jagged1-Notch2 signaling in patterning the bones of the mouse and human middle ear.

Whereas Jagged1-Notch2 signaling is known to pattern the sensorineural components of the inner ear, its role in middle ear development has been less clear. We previously reported a role for Jagged-Notch signaling in shaping skeletal elements derived from the first two pharyngeal arches of zebrafish. Here we show a conserved requirement for Jagged1-Notch2 signaling in patterning the stapes and incus middle ear bones derived from the equivalent pharyngeal arches of mammals. Mice lacking Jagged1 or Notch2 in neural crest-derived cells (NCCs) of the pharyngeal arches display a malformed stapes. Heterozygous Jagged1 knockout mice, a model for Alagille Syndrome (AGS), also display stapes and incus defects. We find that Jagged1-Notch2 signaling functions early to pattern the stapes cartilage template, with stapes malformations correlating with hearing loss across all frequencies. We observe similar stapes defects and hearing loss in one patient with heterozygous JAGGED1 loss, and a diversity of conductive and sensorineural hearing loss in nearly half of AGS patients, many of which carry JAGGED1 mutations. Our findings reveal deep conservation of Jagged1-Notch2 signaling in patterning the pharyngeal arches from fish to mouse to man, despite the very different functions of their skeletal derivatives in jaw support and sound transduction.

A Mouse Model That Reproduces the Developmental Pathways and Site Specificity of the Cancers Associated With the Human BRCA1 Mutation Carrier State.

Predisposition to breast and extrauterine Müllerian carcinomas in BRCA1 mutation carriers is due to a combination of cell-autonomous consequences of BRCA1 inactivation on cell cycle homeostasis superimposed on cell-nonautonomous hormonal factors magnified by the effects of BRCA1 mutations on hormonal changes associated with the menstrual cycle. We used the Müllerian inhibiting substance type 2 receptor (Mis2r) promoter and a truncated form of the Follicle stimulating hormone receptor (Fshr) promoter to introduce conditional knockouts of Brca1 and p53 not only in mouse mammary and Müllerian epithelia, but also in organs that control the estrous cycle. Sixty percent of the double mutant mice developed invasive Müllerian and mammary carcinomas. Mice carrying heterozygous mutations in Brca1 and p53 also developed invasive tumors, albeit at a lesser (30%) rate, in which the wild type alleles were no longer present due to loss of heterozygosity. While mice carrying heterozygous mutations in both genes developed mammary tumors, none of the mice carrying only a heterozygous p53 mutation developed such tumors (P < 0.0001), attesting to a role for Brca1 mutations in tumor development. This mouse model is attractive to investigate cell-nonautonomous mechanisms associated with cancer predisposition in BRCA1 mutation carriers and to investigate the merit of chemo-preventive drugs targeting such mechanisms.

Brca1 Mutations Enhance Mouse Reproductive Functions by Increasing Responsiveness to Male-Derived Scent.

We compared the gene expression profiles of ovarian granulosa cells harboring either mutant or wild type Brca1 to follow up on our earlier observation that absence of a functional Brca1 in these important regulators of menstrual/estrous cycle progression leads to prolongation of the pre-ovulatory phase of the estrous cycle and to increased basal levels of circulating estradiol. Here we show that ovarian granulosa cells from mice carrying a conditional Brca1 gene knockout express substantially higher levels of olfactory receptor mRNA than granulosa cells from wild type littermates. This led us to hypothesize that reproductive functions in mutant female mice might be more sensitive to male-derived scent than in wild type female mice. Indeed, it is well established that isolation from males leads to complete cessation of mouse estrous cycle activity while exposure to olfactory receptor ligands present in male urine leads to resumption of such activity. We found that Brca1-/- female mice rendered anovulatory by unisexual isolation resumed ovulatory activity more rapidly than their wild type littermates when exposed to bedding from cages where males had been housed. The prime mediator of this increased responsiveness appears to be the ovary and not olfactory neurons. This conclusion is supported by the fact that wild type mice in which endogenous ovaries had been replaced by Brca1-deficient ovarian transplants responded to male-derived scent more robustly than mutant mice in which ovaries had been replaced by wild type ovarian transplants. Our findings not only have important implications for our understanding of the influence of olfactory signals on reproductive functions, but also provide insights into mechanisms whereby genetic risk factors for breast and extra uterine Müllerian carcinomas may influence menstrual activity in human, which is itself an independent risk factor for these cancers.

Changes in the mouse estrus cycle in response to BRCA1 inactivation suggest a potential link between risk factors for familial and sporadic ovarian cancer.

Menstrual cycle activity is the most important risk factor for sporadic serous ovarian carcinoma, whereas a germ-line mutation in BRCA1 is the most important risk factor for the familial form. Given the rarity of BRCA1 mutations in sporadic ovarian cancers, we hypothesized that BRCA1 influences the menstrual cycle in a way that mimics the factors underlying sporadic ovarian cancer predisposition, making BRCA1 mutations redundant in such cancers. We compared the length of each phase of the estrus cycle (equivalent to the human menstrual cycle) and of circulating levels of estradiol in control mice and in mice carrying a Brca1 mutation in their ovarian granulosa cells, two thirds of which develop ovarian or uterine epithelial tumors. We also compared the length of the different phases of the cycle in mutants that subsequently developed tumors with those in mutants that remained tumor-free. Mutant mice as well as oophorectomized wild-type mice harboring mutant ovarian grafts showed a relative increase in the average length of the proestrus phase of the estrus cycle, which corresponds to the estrogen-dominated follicular phase of the human menstrual cycle. Total circulating levels of estradiol were also increased in mutant mice injected with pregnant mare serum gonadotropins. The relative increase in proestrus length was highest in mutant mice that subsequently developed reproductive epithelial tumors. We conclude that loss of a functional Brca1 increases murine ovarian epithelial tumor predisposition by increasing estrogen stimulation in the absence of progesterone, recapitulating conditions associated with sporadic ovarian cancer predisposition in humans.

Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest.

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2alpha, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.