Application of full-genome analysis to diagnose rare monogenic disorders.

Current genetic testenhancer and narrows the diagnostic intervals for rare diseases provide a diagnosis in only a modest proportion of cases. The Full-Genome Analysis method, FGA, combines long-range assembly and whole-genome sequencing to detect small variants, structural variants with breakpoint resolution, and phasing. We built a variant prioritization pipeline and tested FGA's utility for diagnosis of rare diseases in a clinical setting. FGA identified structural variants and small variants with an overall diagnostic yield of 40% (20 of 50 cases) and 35% in exome-negative cases (8 of 23 cases), 4 of these were structural variants. FGA detected and mapped structural variants that are missed by short reads, including non-coding duplication, and phased variants across long distances of more than 180 kb. With the prioritization algorithm, longer DNA technologies could replace multiple tests for monogenic disorders and expand the range of variants detected. Our study suggests that genomes produced from technologies like FGA can improve variant detection and provide higher resolution genome maps for future application.

Biallelic variants in the RNA exosome gene EXOSC5 are associated with developmental delays, short stature, cerebellar hypoplasia and motor weakness.

The RNA exosome is an essential ribonuclease complex required for processing and/or degradation of both coding and non-coding RNAs. We identified five patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The clinical features of these patients include failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. While we ascertained five patients, three patients with distinct variants of EXOSC5 were studied in detail. The first patient had a deletion involving exons 5-6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. The additional two patients ascertained are siblings who had an early frameshift mutation in EXOSC5 and the p.Thr114Ile missense variant that were inherited in trans. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess consequences of pathogenic EXOSC5 variants. Loss of function for exosc5 in zebrafish results in shortened and curved tails/bodies, reduced eye/head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants cause defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. These findings expand the number of genes encoding RNA exosome subunits linked to human disease while also suggesting that disease mechanism varies depending on the specific pathogenic variant.

A missense variant, p.(Ile269Asn), in MC4R as a secondary finding in a child with BCL11A-related intellectual disability.

We describe a three year old female who underwent clinical exome sequencing and was diagnosed with BCL11A-related intellectual disability/Dias-Logan syndrome due to a de novo, heterozygous variant in the BCL11A gene, NM_018014.3:c.148C > T; p.(Gln50*). A missense variant in MC4R, NM_005912.3:c.806T > A; p.(Ile269Asn), was also reported as a secondary finding. In her family, her father, paternal aunt, and paternal uncle were all reported to have height and weight measurements suggestive of Class 3 obesity with BMI>40 kg/m2. The MC4R gene is not currently listed among those recommended for reporting of secondary findings by the American College of Medical Genetics and Genomics (ACMG). The identification of genetic risk factors for obesity is an emerging field without established guidelines for the care of patients who are found to have a predisposing genetic variant for obesity as a secondary finding. Management suggestions include interventions for weight-management, early screening for obesity-related co-morbidities, such as diabetes and dyslipidemia, and targeted therapies, such as MC4R agonists.

A novel truncating variant in ring finger protein 113A (RNF113A) confirms the association of this gene with X-linked trichothiodystrophy.

We describe an 11-year old boy with severe global developmental delays, failure to thrive and growth retardation, refractory seizures with recurrent status epilepticus, hypogammaglobulinemia, hypergonadotropic hypogonadism, and duodenal strictures. He had facial and skin findings compatible with trichothiodystrophy, including sparse and brittle hair, thin eyebrows, and dry skin. Exome sequencing showed a hemizygous, truncating variant in RNF113A, c.903_910delGCAGACCA, predicting p.(Gln302fs*12), that was inherited from his mother. Although his clinical features overlap closely with features described in the two previously reported male first cousins with RNF113A loss of function mutations, the duodenal strictures seen in this patient have not been reported. Interestingly, the patient's mother had short stature and 100% skewed X-inactivation as seen in other obligate female carriers. A second male with developmental delays, microcephaly, seizures, ambiguous genitalia, and facial anomalies that included sparse and brittle hair, thin eyebrows and dry skin was recently reported to have c.897_898delTG, predicting p.(Cys299*) in RNF113A and we provide additional clinical details for this patient. This report further supports deleterious variants in RNF113A as a cause of a novel trichothiodystrophy syndrome.

X-linked duplication copy number variation in a familial overgrowth condition.

We describe an overgrowth condition associated with X-linked copy number variation. Three brothers displayed an overgrowth pattern at birth that continued postnatally. Clinical findings included macrocephaly, distinctive facial features, developmental delay and variable clubfoot. Normal fetal growth was noted until the third trimester by Hadlock standards, revealing a late gestational overgrowth pattern. Microarray analysis in the family showed a maternally inherited 680 kb copy number duplication at Xq26.1-q26.2 in all three brothers. Molecular sequencing for known overgrowth conditions including GPC3, Sotos 1 (NSD1), Malan (NFIX), Perlman (DIS3L2), Weaver (EZH2), Opitz-Kaveggia (MED12) loci were negative. BWS IC1 and IC2 methylation and CDKN1C testing was also negative. Normal IGF1 levels excluded X-linked acrogiantism. The duplicated region Xq26.1-q26.2 contained IGSF1 and at least part of the lncRNA FIRRE. IGSF1, a highly expressed pituitary immunoglobulin superfamily gene, was recently implicated in a genome-wide association study of canine size. IGSF1 variants were associated with large canine breeds compared to smaller breeds. Our findings support the hypothesis that an X-linked variant encompassing the IGSF1 region may be associated with body size. Although IGSF1 loss has been noted in human hypothyroidism, this is the first reported phenotype in a family with copy number duplication in the region. Our findings suggest that prenatal evaluation, cross-species evaluation, Mendelian, and GWAS studies may describe a distinctive familial condition and its corresponding phenotypic features.

Schimke immunoosseous dysplasia and management considerations for vascular risks.

Schimke immunoosseous dysplasia (SIOD) is a multisystemic condition characterized by early arteriosclerosis and progressive renal insufficiency, among other features. Many SIOD patients have severe, migraine-like headaches, transient neurologic attacks, or cerebral ischemic events. Cerebral events could be exacerbated or precipitated by hypertension, and it is unclear how these are related to arteriosclerotic changes as dyslipidemia is also a feature of SIOD. The correlation between hypercholesterolemia and cardiovascular risk in SIOD is unclear. Also, the etiology and management of headaches is not well characterized. Here we report our clinical observations in the management of SIOD in a patient who was diagnosed in school age despite early signs and symptoms. We describe biallelic variants, including a previously unreported c.1931G>A (p.Arg644Gln) variant in SMARCAL1. We specifically investigated whether migraine-like headaches and progressive nephropathy may be related to blood pressure dysregulation. We found a correlation between tighter blood pressure regulation using ambulatory blood pressure monitoring and a subjective decrease in headache symptoms. We discuss blood pressure medication management in SIOD. We also characterize dyslipidemia relative to atherosclerosis risks and provide new management strategies to consider for optimizing care.

Extracutaneous manifestations in phacomatosis cesioflammea and cesiomarmorata: Case series and literature review.

Phacomatosis pigmentovascularis (PPV) comprises a family of rare conditions that feature vascular abnormalities and melanocytic lesions that can be solely cutaneous or multisystem in nature. Recently published work has demonstrated that both vascular and melanocytic abnormalities in PPV of the cesioflammea and cesiomarmorata subtypes can result from identical somatic mosaic activating mutations in the genes GNAQ and GNA11. Here, we present three new cases of PPV with features of the cesioflammea and/or cesiomarmorata subtypes and mosaic mutations in GNAQ or GNA11. To better understand the risk of potentially occult complications faced by such patients we additionally reviewed 176 cases published in the literature. We report the frequency of clinical findings, their patterns of co-occurrence as well as published recommendations for surveillance after diagnosis. Features assessed include: capillary malformation; dermal and ocular melanocytosis; glaucoma; limb asymmetry; venous malformations; and central nervous system (CNS) anomalies, such as ventriculomegaly and calcifications. We found that ocular findings are common in patients with phacomatosis cesioflammea and cesiomarmorata. Facial vascular involvement correlates with a higher risk of seizures (p = .0066). Our genetic results confirm the role of mosaic somatic mutations in GNAQ and GNA11 in phacomatosis cesioflammea and cesiomarmorata. Their clinical and molecular findings place these conditions on a clinical spectrum encompassing other GNAQ and GNA11 related disorders and inform recommendations for their management.

Familial melanoma-astrocytoma syndrome: synchronous diffuse astrocytoma and pleomorphic xanthoastrocytoma in a patient with germline CDKN2A/B deletion and a significant family history.

Familial melanoma-astrocytoma syndrome is a tumor predisposition syndrome caused by inactivating germline alteration of the CDKN2A tumor suppressor gene on chromosome 9p21. While some families with germline CDKN2A mutations are prone to development of just melanomas, other families develop both melanomas, astrocytomas, and occasionally other nervous-system neoplasms including peripheral nerve sheath tumors and meningiomas. The histologic spectrum of the astrocytomas that arise as part of this syndrome is not well described, nor are the additional genetic alterations that drive these astrocytomas apart from the germline CDKN2A inactivation. Herein, we report the case of a young man with synchronous development of a pleomorphic xanthoastrocytoma, diffuse astrocytoma, and paraspinal mass radiographically consistent with a peripheral nerve sheath tumor. His paternal family history is significant for melanoma, glioblastoma, and oral squamous cell carcinoma. Genomic profiling revealed that he harbors a heterozygous deletion in the germline of chromosome 9p21.3 encompassing the CDKN2A and CDKN2B tumor suppressor genes. Both the pleomorphic xanthoastrocytoma and diffuse astrocytoma were found to have homozygous deletion of CDKN2A/B due to somatic loss of the other copy of chromosome 9p containing the remaining intact alleles. Additional somatic alterations included BRAF p.V600E mutation in the pleomorphic xanthoastrocytoma and PTPN11, ATRX, and NF1 mutations in the diffuse astrocytoma. The presence of germline CDKN2A/B inactivation together with the presence of multiple anatomically, histologically, and genetically distinct astrocytic neoplasms, both with accompanying somatic loss of heterozygosity for the CDKN2A/B deletion, led to a diagnosis of familial melanoma-astrocytoma syndrome. This remarkable case illustrates the histologic and genetic diversity that astrocytomas arising as part of this rare glioma predisposition syndrome can demonstrate.
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Role of heparan sulfatases in ovarian and breast cancer.

Endosulfatases HSulf-1 and -2 (also referred to as Sulf1 and -2) represent a family of enzymes that modulate heparin binding growth factor signaling. Heparan sulfatase 1 (HSulf-1) and heparan sulfatase 2 (HSulf-2) are two important 6-O endosulfatases which remove or edit 6-O sulfate residues of N-glucosamine present on highly sulfated HS. Alteration of heparan sulfatases have been identified in the context of several cancer types. Many cancer types either exhibit increased or decreased HSulfs expression at the transcript levels. Specifically, HSulf-1 was found to be downregulated in early-stage ovarian tumors, hepatocellular carcinoma, and metastatic breast cancer patients. HSulf-2 was found to be upregulated in ductal carcinoma in situ and invasive ductal carcinoma, whereas limited information is present about HSulf-2 expression in different stages of ovarian cancers. Here, we review the important role of these sulfatases play in ovarian and breast cancers in terms of tumorigenesis such as angiogenesis, chemoresistance, apoptosis, growth factor signaling, hypoxia and metastasis. These recent discoveries have added significant understanding about these sulfate editing enzymes.

Cleavage of PGRP-LC receptor in the Drosophila IMD pathway in response to live bacterial infection in S2 cells.

Drosophila responds to Gram-negative bacterial infection by activating the immune deficiency (IMD) pathway, leading to production of antimicrobial peptides (AMPs). As a receptor for the IMD pathway, peptidoglycan-recognition protein (PGRP), PGRP-LC is known to recognize and bind monomeric peptidoglycan (DAP-type PGN) through its PGRP ectodomain and in turn activate the IMD pathway. The questions remain how PGRP-LC is activated in response to pathogen infection to initiate the IMD signal transduction in Drosophila. Here we present evidence to show that proteases such as elastase and Mmp2 can also activate the IMD pathway but not the TOLL pathway. The elastase-dependent IMD activation requires the receptor PGRP-LC. Importantly, we find that live Salmonella/E. coli infection modulates PGRP-LC expression/receptor integrity and activates the IMD pathway while dead Salmonella/E. coli or protease-deficient E. coli do neither. Our results suggest an interesting possibility that Gram-negative pathogen infection may be partially monitored through the structural integrity of the receptor PGRP-LC via an infection-induced enzyme-based cleavage-mediated activation mechanism.

High temperature requirement A3 (HtrA3) promotes etoposide- and cisplatin-induced cytotoxicity in lung cancer cell lines.

Lung cancer is the leading cause of cancer-related deaths worldwide. Here we show for the first time that HtrA3 is a mitochondrial stress-response factor that promotes cytotoxicity to etoposide and cisplatin in lung cancer cell lines. Exogenous expression of wild type HtrA3 domain variants significantly attenuated cell survival with etoposide and cisplatin treatment in lung cancer cell lines H157 and A549 compared with expression of protease inactive mutants (S305A) or vector control. Conversely, HtrA3 suppression promoted cell survival with etoposide and cisplatin treatment in lung cancer cell lines Hop62 and HCC827. Survival was attenuated by re-expression of wild type HtrA3 variants during treatment but not by protease inactive mutants or vector control. HtrA3 also co-fractionated and co-localized with mitochondrial markers with both endogenous and exogenous expression in normal lung and lung cancer cell lines but was translocated from mitochondria following etoposide treatment. Moreover, HtrA3 translocation from mitochondria correlated with an increase in cell death that was attenuated by either HtrA3 suppression or Bcl-2 overexpression. Taken together, these results suggest that HtrA3 may be a previously uncharacterized mitochondrial cell death effector whose serine protease function may be crucial to modulating etoposide- and cisplatin-induced cytotoxicity in lung cancer cell lines.

Methylation induced gene silencing of HtrA3 in smoking-related lung cancer.

PURPOSE: Some 85% of lung cancers are smoking related. Here, we investigate the role of serine protease HtrA3 in smoking-related lung cancer. EXPERIMENTAL DESIGN: We assess HtrA3 methylation and its corresponding expression in the human bronchial cell line BEAS-2B following cigarette smoke carcinogen treatment, in lung cancer cell lines and in primary lung tumors from light, moderate, and heavy smokers. We also show the effects of HtrA3 downregulation on MTT reduction and clonogenic survival with etoposide and cisplatin treatment and the corresponding effects of HtrA3 re-expression during treatment. RESULTS: We show for the first time that HtrA3 expression is reduced or completely lost in over 50% of lung cancer cell lines and primary lung tumors from heavy smokers. Treatment of HtrA3-deficient cell lines with 5-aza-2'-deoxycytidine resulted in a dose-dependent increase in HtrA3 transcription. Further, sequence analysis of bisulfite-modified DNA from lung cancer cell lines and from primary lung tumors showed an increased frequency of methylation within the first exon of HtrA3 with a corresponding loss of HtrA3 expression, particularly in tumors from smokers. In BEAS-2B, treatment with the cigarette smoke carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone resulted in HtrA3 downregulation with a corresponding increase in methylation. Additional studies indicate resistance to etoposide and cisplatin cytotoxicity as a functional consequence of HtrA3 loss. Finally, immunohistochemical analysis of primary lung tumors revealed a strong correlation between low HtrA3 expression and heavy smoking history. CONCLUSIONS: Collectively, these results suggest that cigarette smoke-induced methylation of HtrA3 could contribute to the etiology of chemoresistant disease in smoking-related lung cancer.

Cell proliferation and epidermal growth factor signaling in non-small cell lung adenocarcinoma cell lines are dependent on Rin1.

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Delta) complemented the Rin1 depletion effects, and overexpression of Rin1Delta had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.