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Supplementary Figures 1-4. Supplementary Figure S1: Full Pedigrees from TP53 c.1000G>C;p.G334R clinically ascertained families. Fam ID allows reference to Table 1; Supplementary Figure S2: Constructs encoding p53 wild-type (TP53-WT), p53-R175H (TP53-R175H), and p53-G334R (c.1000G>A and c.1000G>C) were transiently transfected into p53 deficient Saos-2 cells; Supplementary Figure S3: p53 levels after treatment with 10uM cisplatin (CDDP); Supplementary Figure S4: Heatmap showing fold activation of a canonical p53 signature at 24 hours post nutlin with wildtype versus mutant lymphoblastoid cell lines.

Germline mutations in TP53 cause a rare high penetrance cancer syndrome, Li–Fraumeni syndrome (LFS). Here, we identified a rare TP53 tetramerization domain missense mutation, c.1000G>C;p.G334R, in a family with multiple late-onset LFS-spectrum cancers. Twenty additional c.1000G>C probands and one c.1000G>A proband were identified, and available tumors showed biallelic somatic inactivation of TP53. The majority of families were of Ashkenazi Jewish descent, and the TP53 c.1000G>C allele was found on a commonly inherited chromosome 17p13.1 haplotype. Transient transfection of the p.G334R allele conferred a mild defect in colony suppression assays. Lymphoblastoid cell lines from the index family in comparison with TP53 normal lines showed that although classical p53 target gene activation was maintained, a subset of p53 target genes (including PCLO, PLTP, PLXNB3, and LCN15) showed defective transactivation when treated with Nutlin-3a. Structural analysis demonstrated thermal instability of the G334R-mutant tetramer, and the G334R-mutant protein showed increased preponderance of mutant conformation. Clinical case review in comparison with classic LFS cohorts demonstrated similar rates of pediatric adrenocortical tumors and other LFS component cancers, but the latter at significantly later ages of onset. Our data show that TP53 c.1000G>C;p.G334R is found predominantly in Ashkenazi Jewish individuals, causes a mild defect in p53 function, and leads to low penetrance LFS.Significance:TP53 c.1000C>G;p.G334R is a pathogenic, Ashkenazi Jewish–predominant mutation associated with a familial multiple cancer syndrome in which carriers should undergo screening and preventive measures to reduce cancer risk.

Contains fulltext : 248942.pdf (Publisher’s version ) (Open Access) The caudal type homeobox 2 (CDX2) gene encodes a developmental regulator involved in caudal body patterning. Only three pathogenic variants in human CDX2 have been described, in patients with persistent cloaca, sirenomelia and/or renal and anogenital malformations. We identified five patients with de novo or inherited pathogenic variants in CDX2 with clinical phenotypes that partially overlap with previous cases, that is, imperforate anus and renal, urogenital and limb abnormalities. However, additional clinical features were seen including vertebral agenesis and we describe considerable phenotypic variability, even in unrelated patients with the same recurrent p.(Arg237His) variant. We propose CDX2 variants as rare genetic cause for a multiple congenital anomaly syndrome that can include features of caudal regression syndrome and VACTERL. A causative role is further substantiated by the relationship between CDX2 and other proteins encoded by genes that were previously linked to caudal abnormalities in humans, for example, TBXT (sacral agenesis and other vertebral segmentation defects) and CDX1 (anorectal malformations). Our findings confirm the essential role of CDX2 in caudal morphogenesis and formation of cloacal derivatives in humans, which to date has only been well characterized in animals.

BACKGROUND AND OBJECTIVES Telemedicine may increase access to medical genetics care. However, in the pediatric setting, how telemedicine may affect the diagnostic rate is unknown, partially because of the perceived importance of the dysmorphology physical examination. We studied the clinical effectiveness of telemedicine for patients with suspected or confirmed genetic conditions. METHODS We conducted a retrospective cohort study of outpatient encounters before and after the widespread implementation of telemedicine (N = 5854). Visit types, diagnoses, patient demographic characteristics, and laboratory data were acquired from the electronic health record. Patient satisfaction was assessed through survey responses. New molecular diagnosis was the primary end point. RESULTS Patients seen by telemedicine were more likely to report non-Hispanic White ancestry, prefer to speak English, live in zip codes with higher median incomes, and have commercial insurance (all P < .01). Genetic testing was recommended for more patients evaluated by telemedicine than in person (79.5% vs 70.9%; P < .001). Patients seen in person were more likely to have a sample collected, resulting in similar test completion rates (telemedicine, 51.2%; in person, 55.1%; P = .09). There was no significant difference in molecular diagnosis rate between visit modalities (telemedicine, 13.8%; in person, 12.4%; P = .40). CONCLUSIONS Telemedicine and traditional in-person evaluation resulted in similar molecular diagnosis rates. However, improved methodologies for remote sample collection may be required. This study reveals the feasibility of telemedicine in a large academic medical genetics practice and is applicable to other pediatric specialties with perceived importance of physical examination.

The semaphorin protein family is a diverse set of extracellular signaling proteins that perform fundamental roles in the development and operation of numerous biological systems, notably the nervous, musculoskeletal, cardiovascular, endocrine, and reproductive systems. Recently, recessive loss-of-function (LoF) variants in SEMA3A (semaphorin 3A) have been shown to result in a recognizable syndrome characterized by short stature, skeletal abnormalities, congenital heart defects, and variable additional anomalies. Here, we describe the clinical and molecular characterization of a female patient presenting with skeletal dysplasia, hypogonadotropic hypogonadism (HH), and anosmia who harbors a nonsense variant c.1633C>T (p.Arg555*) and a deletion of exons 15, 16, and 17 in SEMA3A in the compound heterozygous state. These variants were identified through next-generation sequencing analysis of a panel of 26 genes known to be associated with HH/Kallmann syndrome. Our findings further substantiate the notion that biallelic LoF SEMA3A variants cause a syndromic form of short stature and expand the phenotypic spectrum associated with this condition to include features of Kallmann syndrome.

Background Several studies have reported conflicting evidence on the inclusion of testicular germ cell tumors (TGCT) in the DICER1 tumor-predisposition phenotype. We evaluated the relationship between DICER1 and TGCT by reviewing scrotal ultrasounds of males with pathogenic germline variants in DICER1 and queried exome data from TGCT-affected men for DICER1 variants. Methodology Fifty-four male DICER1-carriers and family controls (n=41) enrolled in the National Cancer Institute (NCI) DICER1 Natural History Study were offered scrotal ultrasounds. These studies were examined by a single radiologist for abnormalities. In parallel, DICER1 variants from two large exome-sequenced TGCT cohorts were extracted. We used previously published AMG-AMP criteria to characterize rare DICER1 variants. Results There was no observed difference in frequency of testicular cystic structures in DICER1-carriers versus controls. DICER1 variation was not associated with TGCT in the NCI DICER1-carriers. In 1,264 exome-sequenced men with TGCT, none harbored ClinVar- or InterVar-determined pathogenic or likely pathogenic variants in DICER1. Three DICER1 variants of uncertain significance (one case and two controls) were predicted “damaging” based on a priori criteria. Conclusion Using two complementary approaches, we found no evidence of an association between pathogenic DICER1 variants and TGCT.

Germline mutations in TP53 cause a rare high penetrance cancer syndrome, Li–Fraumeni syndrome (LFS). Here, we identified a rare TP53 tetramerization domain missense mutation, c.1000G>C;p.G334R, in a family with multiple late-onset LFS-spectrum cancers. Twenty additional c.1000G>C probands and one c.1000G>A proband were identified, and available tumors showed biallelic somatic inactivation of TP53. The majority of families were of Ashkenazi Jewish descent, and the TP53 c.1000G>C allele was found on a commonly inherited chromosome 17p13.1 haplotype. Transient transfection of the p.G334R allele conferred a mild defect in colony suppression assays. Lymphoblastoid cell lines from the index family in comparison with TP53 normal lines showed that although classical p53 target gene activation was maintained, a subset of p53 target genes (including PCLO, PLTP, PLXNB3, and LCN15) showed defective transactivation when treated with Nutlin-3a. Structural analysis demonstrated thermal instability of the G334R-mutant tetramer, and the G334R-mutant protein showed increased preponderance of mutant conformation. Clinical case review in comparison with classic LFS cohorts demonstrated similar rates of pediatric adrenocortical tumors and other LFS component cancers, but the latter at significantly later ages of onset. Our data show that TP53 c.1000G>C;p.G334R is found predominantly in Ashkenazi Jewish individuals, causes a mild defect in p53 function, and leads to low penetrance LFS. Significance: TP53 c.1000C>G;p.G334R is a pathogenic, Ashkenazi Jewish–predominant mutation associated with a familial multiple cancer syndrome in which carriers should undergo screening and preventive measures to reduce cancer risk.

Teebi hypertelorism syndrome (THS; OMIM 145420) is a rare craniofacial disorder characterized by hypertelorism, prominent forehead, short nose with broad or depressed nasal root. Some cases of THS have been attributed to SPECC1L variants. Homozygous variants in CDH11 truncating the transmembrane and intracellular domains have been implicated in Elsahy-Waters syndrome (EWS; OMIM 211380) with hypertelorism. We report THS due to CDH11 heterozygous missense variants on 19 subjects from 9 families. All affected residues in the extracellular region of Cadherin-11 (CHD11) are highly conserved across vertebrate species and classical cadherins. Six of the variants that cluster around the EC2-EC3 and EC3-EC4 linker regions are predicted to affect Ca2+ binding that is required for cadherin stability. Two of the additional variants [c.164G > C, p.(Trp55Ser) and c.418G > A, p.(Glu140Lys)] are also notable as they are predicted to directly affect trans-homodimer formation. Immunohistochemical study demonstrates that CDH11 is strongly expressed in human facial mesenchyme. Using multiple functional assays, we show that five variants from the EC1, EC2-EC3 linker, and EC3 regions significantly reduced the cell-substrate trans adhesion activity and one variant from EC3-EC4 linker results in changes in cell morphology, focal adhesion, and migration, suggesting dominant negative effect. Characteristic features in this cohort included depressed nasal root, cardiac and umbilical defects. These features distinguished this phenotype from that seen in SPECC1L-related hypertelorism syndrome and CDH11-related EWS. Our results demonstrate heterozygous variants in CDH11, which decrease cell-cell adhesion and increase cell migratory behavior, cause a form of THS, as termed CDH11-related THS.

Testicular germ cell tumors (TGCT) are the most common tumor in young white men and have a high heritability. In this study, the international Testicular Cancer Consortium assemble 10,156 and 179,683 men with and without TGCT, respectively, for a genome-wide association study. This meta-analysis identifies 22 TGCT susceptibility loci, bringing the total to 78, which account for 44% of disease heritability. Men with a polygenic risk score (PRS) in the 95th percentile have a 6.8-fold increased risk of TGCT compared to men with median scores. Among men with independent TGCT risk factors such as cryptorchidism, the PRS may guide screening decisions with the goal of reducing treatment-related complications causing long-term morbidity in survivors. These findings emphasize the interconnected nature of two known pathways that promote TGCT susceptibility: male germ cell development within its somatic niche and regulation of chromosomal division and structure, and implicate an additional biological pathway, mRNA translation., Testicular germ cell tumors are highly heritable, and the authors present the largest genome association study, identifying 22 novel loci, which account for a third of those identified to date. Implicated pathways include male germ cell development and differentiation, and chromosomal segregation.

A male infant is born at 38 weeks 3 days of gestational age to a gravida 5, para 1 woman via spontaneous vaginal delivery, after a pregnancy complicated by maternal preeclampsia. Maternal history is significant for 2 early miscarriages and an ectopic pregnancy. All prenatal laboratory findings, including maternal immunoglobulin G for herpes simplex virus (HSV), had been normal or negative. The family history is unremarkable. The infant requires normal newborn care and is discharged from the hospital on day 2 after birth. His parents note that he had been sleepy and a poor feeder. He is hypothermic and tachypneic at his first newborn visit to the pediatrician and is immediately referred to the emergency department. Septic evaluation reveals HSV in both serum and cerebrospinal fluid. Ammonia concentration is normal and his complete blood cell count reveals leukopenia. Initial treatment includes acyclovir, cefotaxime, and ampicillin, narrowed within 48 hours to acyclovir monotherapy. His condition rapidly declines and he develops respiratory failure on hospital day (HOD) 1. The ammonia concentration is within the normal range (39 μg/dL [28 μmol/L]). Feeding is initiated on HOD 2 with a subsequent rise in the ammonia concentration (113 μg/dL [81 μmol/L]). Despite the cessation of feeding and initiation of moderate glucose infusion rate, the ammonia concentration rises to 538 μg/dL (384 μmol/L) by HOD 4. The patient is transferred to a different hospital for metabolic consultation. Increased glucose …

OBJECTIVE: To evaluate the odds of a behavioral health diagnosis among youth with a difference of sex development (DSD) or congenital adrenal hyperplasia (CAH) compared with matched controls in the PEDSnet database. STUDY DESIGN: All youth with a diagnosis of DSD (n=1,216) or CAH (n=1,647) and at least one outpatient encounter were extracted from the PEDSnet database and propensity-score matched on 8 variables (1:4) to controls (n=4,864 and 6,588, respectively) using multivariable logistic regression. The likelihood of having behavioral health diagnoses was examined using generalized estimating equations. RESULTS: Youth with a DSD had higher odds of a behavioral health diagnosis (OR: 1.7 [95% CI: 1.4, 2.1], p

We present 2 patients found to have ovotesticular disorder of sexual development (otDSD) in late adolescence. Two 15-year-old phenotypically male patients presented to a large pediatric hospital with different complaints: 1 with concern for testicular rupture after a straddle injury; 1 with gynecomastia. Further workup, including imaging and laboratory tests, was performed before surgical exploration. The first patient had unilateral ovotestis, contralateral testis, and SRY-negative 46,XX karyotype. The second patient with gynecomastia had unilateral ovotestis with hemi-uterus and fallopian tube, contralateral ovarian tissue, and 46,XX/47,XXY Klinefelter mosaic karyotype. Although rare, phenotypically normal male patients may present later with ovotesticular disorder of sexual development.

Differences of Sex Development (DSD) includes a wide spectrum of etiologies and phenotypes. A subset of individuals with DSDs are predisposed to gonadal germ cell tumor (GCT). In this setting, GCT risk varies widely, depending on the DSD molecular etiology and penetrance. Prognostication based on molecular diagnosis remains challenging, as natural history data specific to recently identified molecular causes of DSD is lacking. In this review, we provide a framework for the clinical geneticist to consider GCT tumor risk in the patient with DSD. We discuss germ cell development and etiology of GCT growth, along with parameters to consider when recommending prophylactic gonadectomy including fertility, hormonal output, and malignant GTC treatment outcomes. Shortly after the 2006 reorganization of DSD nomenclature, literature reviews of natural history publications stratified GCT risk by a chromosomal, pathological, and hormonal taxonomy. Our 2017 literature review reveals a larger body of publications. However, the broad DSD GCT risk stratification within the 2006 taxonomy remains stable. We discuss precise GCT risk assessment for specific diagnoses, including androgen insensitivity, Smith-Lemli-Opitz, and 46,XY with MAP3K1 mutations and gonadal dysgenesis, as examples. We also examine the GCT risk in non-DSD syndromes, in addition to the cancer risks in DSD patients with dimorphic gonads and genitalia. This review is intended to provide a nuanced assessment of relative germ cell tumor risk in the DSD patient, including modern precise molecular diagnosis, for use by the clinical geneticist.

In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.

Myelin Regulatory Factor (MYRF) is a transcription factor that has previously been associated with the control of the expression of myelin-related genes. However, it is highly expressed in human tissues and mouse embryonic tissues outside the nervous system such as the stomach, lung, and small intestine. It has not previously been reported as a cause of any Mendelian disease. We report here two males with Scimitar syndrome [MIM 106700], and other features including penoscrotal hypospadias, cryptorchidism, pulmonary hypoplasia, tracheal anomalies, congenital diaphragmatic hernia, cleft spleen, thymic involution, and thyroid fibrosis. Gross neurologic functioning appears to be within normal limits. In both individuals a de novo variant in MYRF was identified using exome sequencing. Neither variant is found in gnomAD. Heterozygous variants in MYRF should be considered in patients with variants of Scimitar syndrome and urogenital anomalies.

Testicular germ cell tumors (TGCT) are ideally suited to agnostic genome-wide association methods because of their known high heritability and homogeneous cell of origin. Here we report the most updated results from the international Testicular Cancer Consortium (TECAC) that extends published findings from our recent meta-analysis, which included 3,558 cases and 13,970 controls from five centers. To these existing samples, we added summary statistics from 300 cases and 151,991 controls provided by deCODE combined with imputed genotypes from an additional 5,602 cases and 5,006 controls assembled from 15 TECAC centers, almost all (97%) of which were centrally genotyped using the Illumina Human Core array. A total of 9,458 TGCT cases were included in our current meta-analysis, representing a 66% increase since our last publication. We identified 14 new and confirmed 41 previously reported loci that surpassed genome-wide significance (5 × 10-8). Fifteen previously reported loci did not reach this threshold, although one did so in subgroup analysis. In addition, 57 new loci attained nominal significance (5 × 10-8 ≤ p ≤ 1 × 10-5). As expected, a substantial proportion of top hits continue to map to genetic regions belonging to biological pathways essential to male germ cell development, sex determination, and chromosomal segregation. For example, newly identified genes include anaphase promoting complex subunit 2 (ANAPC2), BCL2 like 11 (BCL211), anti-Mullerian hormone receptor type 2 (AMHR2), and DEP domain containing mTOR interacting protein (DEPTOR). Our findings also further implicate genes located on the X chromosome, specifically the androgen receptor (AR). In analyses stratified by tumor subtype, we identified GATA-binding protein 4 (GATA4) as a susceptibility locus among men diagnosed with seminoma. Despite a similar effect size observed among men diagnosed solely with non-seminoma (i.e. excluding cases with a mixture of both seminoma and non-seminoma tumors), this marker did not surpass genome-wide significance in this subtype likely because of the smaller case sample size. GATA4 previously has been associated with overall risk of TGCT (Litchfield et al., Nat Genet. 2017). Biological mechanisms for all genome-wide significant loci are currently being explored using publicly available data sets, ATAC-seq of four TGCT cell lines, and SPATIaL-Seq (a novel high-resolution chromatin conformation capture (3C) assay) of the NTERA2 pluripotent human embryonal carcinoma cell line. Additional genotyping of top hits in an independent sample set of 1,103 TGCT cases and 1,210 controls is currently ongoing. Citation Format: Louise C. Pyle, John Pluta, Kevin T. Nead, Nandita Mitra, Javier Benitez, D. Timothy Bishop, Victoria Cortessis, Alberto Ferlin, Jourik Gietema, Mark Greene, Tom Grotmol, Ramneek Gupta, Rob Hamilton, Michelle A. Hildebrandt, Trine B. Haugen, Lambertus Kiemeney, Christian Kubisch, Davor Lessel, Paloma Martin, Thorunn Rafnar, Lorenzo Richiardi, Rolf Skotheim, Clare Turnbull, Fredrik Wiklund, Tongzhang Zheng, Ewa Rajpert-De Meyts, Stephen M. Schwartz, Katherine A. McGlynn, Peter A. Kanetsky, Katherine L. Nathanson, Testicular Cancer Consortium (TECAC). Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2684.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development., Author summary Hyperopia or farsightedness is a common condition that can cause visual impairment especially in children. The extreme of this condition is called nanophthalmos, a small crowded eye in which inappropriate drainage of aqueous humor from the eye can lead to glaucoma and vision loss. We previously described a large family with inherited nanophthalmos, but the genetic defect that segregated in this family was unknown. Here, we have used a new approach combining linkage analysis and pooled sequencing to identify the genetic cause in this family. We identified a splice site mutation that causes the myelin regulatory factor (MYRF) gene to produce an aberrant protein. Additionally, a child with syndromic manifestations and a deleterious MYRF variant shared the same eye condition. Using a mouse model in which MYRF is absent from eye tissue during early development, we established a role for this transcription factor in the development of the retinal pigment epithelium and retina. We showed that MYRF interacts with and regulates expression of another membrane protein, TMEM98, which has been implicated in nanophthalmos. Our study establishes MYRF as a new disease gene for nanophthalmos and a regulator of eye development.

IMPORTANCE Approximately 50% of the risk for the development of testicular germ cell tumors (TGCTs) is estimated to be heritable, but no mendelian TGCT predisposition genes have yet been identified. It is hypothesized that inherited pathogenic DNA repair gene (DRG) alterations may drive susceptibility to TGCTs. OBJECTIVE To systematically evaluate the enrichment of germline pathogenic variants in the mendelian cancer predisposition DRGs in patients with TGCTs vs healthy controls. DESIGN, SETTING, AND PARTICIPANTS A case-control enrichment analysis was performed from January 2016 to May 2018 to screen for 48 DRGs in 205 unselected men with TGCT and 27 173 ancestry-matched cancer-free individuals from the Exome Aggregation Consortium cohort in the discovery stage. Significant findings were selectively replicated in independent cohorts of 448 unselected men with TGCTs and 442 population-matched controls, as well as 231 high-risk men with TGCTs and 3090 ancestry- matched controls. Statistical analysis took place from January to May 2018. MAIN OUTCOMES AND MEASURES Gene-level enrichment analysis of germline pathogenic variants in individuals with TGCTs relative to cancer-free controls. RESULTS Among 205 unselected men with TGCTs (mean [SD] age, 33.04 [9.67] years), 22 pathogenic germline DRG variants, one-third of which were in CHEK2 (OMIM 604373), were identified in 20 men (9.8% ; 95% CI, 6.1%-14.7%). Unselected men with TGCTs were approximately 4 times more likely to carry germline loss-of-function CHEK2 variants compared with cancer-free individuals from the Exome Aggregation Consortium cohort (odds ratio [OR], 3.87 ; 95% CI, 1.65-8.86 ; nominal P = .006 ; q = 0.018). Similar enrichment was also seen in an independent cohort of 448 unselected Croatian men with TGCTs (mean [SD] age, 31.98 [8.11] years) vs 442 unselected Croatian men without TGCTs (at least 50 years of age at time of sample collection) (OR, >1.4 ; P = .03) and 231 high-risk men with TGCTs (mean [SD] age, 31.54 [9.24] years) vs 3090 men (all older than 50 years) from the Penn Medicine Biobank (OR, 6.30 ; 95% CI, 2.34- 17.31 ; P = .001). The low-penetrance CHEK2 variant (p.Ile157Thr) was found to be a Croatian founder TGCT risk variant (OR, 3.93 ; 95% CI, 1.53-9.95 ; P = .002). Individuals with the pathogenic CHEK2 loss-of-function variants developed TGCTs 6 years earlier than individuals with CHEK2 wild-type alleles (5.95 years ; 95% CI, 1.48-10.42 ; P = .009). CONCLUSIONS AND RELEVANCE This multicenter case-control analysis of men with or without TGCTs provides evidence for CHEK2 as a novel moderate-penetrance TGCT susceptibility gene, with potential clinical utility. In addition to highlighting DNA-repair deficiency as a potential mechanism driving TGCT susceptibility, this analysis also provides new avenues to explore management strategies and biological investigations for high-risk individuals.

Despite greatly improved morbidity and mortality among infants with medium-chain acyl-CoA dehydrogenase deficiency (MCAD) since the implementation of universal newborn screening (NBS), a population of neonates still becomes ill before their positive screen results are available. Exclusive breastfeeding is a proposed risk factor in this group. Since initial studies of MCAD NBS, breastfeeding rates have increased substantially. In this study, we quantify the current risk of early decompensation in neonates with MCAD and identify factors associated with poor outcomes. We completed a retrospective analysis of neonates with MCAD referred to our center between 2010 and 2015. Of 46 infants with MCAD, 11 (23.9%) were symptomatic before the return of the NBS results. Four died or had cardiac arrest; the remaining seven had lethargy and hypoglycemia. All symptomatic patients were exclusively breastfed; only 40.6% of asymptomatic patients were exclusively breastfed. Breastfeeding rates increased from 45.5% in 2010–2011 to 64.7% in 2012–2013 and 87.5% in 2014–2015. Over these same periods, rates of early decompensation increased from 9.09% to 23.5% and 75%, respectively. Exclusively breastfed neonates with MCAD are at risk for early metabolic decompensation. As breastfeeding rates increase, close management of feeding difficulties is essential for all neonates awaiting NBS results. Genet Med 18 12, 1315–1319.

Mandibulofacial dysostosis with microcephaly (MFDM) is a multiple malformation syndrome comprising microcephaly, craniofacial anomalies, hearing loss, dysmorphic features, and, in some cases, esophageal atresia. Haploinsufficiency of a spliceosomal GTPase, U5-116 kDa/EFTUD2, is responsible. Here, we review the molecular basis of MFDM in the 69 individuals described to date, and report mutations in 38 new individuals, bringing the total number of reported individuals to 107 individuals from 94 kindreds. Pathogenic EFTUD2 variants comprise 76 distinct mutations and seven microdeletions. Among point mutations, missense substitutions are infrequent (14 out of 76; 18%) relative to stop-gain (29 out of 76; 38%), and splicing (33 out of 76; 43%) mutations. Where known, mutation origin was de novo in 48 out of 64 individuals (75%), dominantly inherited in 12 out of 64 (19%), and due to proven germline mosaicism in four out of 64 (6%). Highly penetrant clinical features include, microcephaly, first and second arch craniofacial malformations, and hearing loss; esophageal atresia is present in an estimated ∼27%. Microcephaly is virtually universal in childhood, with some adults exhibiting late "catch-up" growth and normocephaly at maturity. Occasionally reported anomalies, include vestibular and ossicular malformations, reduced mouth opening, atrophy of cerebral white matter, structural brain malformations, and epibulbar dermoid. All reported EFTUD2 mutations can be found in the EFTUD2 mutation database (http://databases.lovd.nl/shared/genes/EFTUD2).

Discordance between clinical phenotype and genotype has multiple causes, including mosaicism. Phenotypes can be modified due to tissue distribution, or the presence of multiple abnormal cell lines with different genomic contributions. We have studied a 20-month-old female whose main phenotypes were failure to thrive, developmental delay, and patchy skin pigmentation. Initial chromosome and SNP microarray analysis of her blood revealed a non-mosaic ∼24 Mb duplication of 15q25.1q26.3 resulting from the unbalanced translocation of terminal 15q to the short arm of chromosome 15. The most common feature associated with distal trisomy 15q is prenatal and postnatal overgrowth, which was not consistent with this patient's phenotype. The phenotypic discordance, in combination with the patchy skin pigmentation, suggested the presence of mosaicism. Further analysis of skin biopsies from both hyper- and hypopigmented regions confirmed the presence of an additional cell line with the short arm of chromosome X deleted and replaced by the entire long arm of chromosome 15. The Xp deletion, consistent with a variant Turner Syndrome diagnosis, better explained the patient's phenotype. Parental studies revealed that the alterations in both cell lines were de novo and the duplicated distal 15q and the deleted Xp were from different parental origins, suggesting a mitotic event. The possible mechanism for the occurrence of two mutually exclusive structural rearrangements with both involving the long arm of chromosome 15 is discussed.

Variants in the SLC25A3 gene, which codes for the mitochondrial phosphate transporter (PiC), lead to a failure of inorganic phosphate (Pi) transport across the mitochondrial membrane, which is required in the final step of oxidative phosphorylation. The literature described two affected sibships with variants in SLC25A3; all cases had skeletal myopathy and cardiomyopathy (OMIM 610773). We report here two new patients who had neonatal cardiomyopathy; one of whom did not have skeletal myopathy nor elevated lactate. Patient 1 had a homozygous splice site variant, c.158-9A>G, which has been previously reported in a Turkish family. Patient 2 was found to be a compound heterozygote for two novel variants, c.599T>G (p.Leu200Trp) and c. 886_898delGGTAGCAGTGCTTinsCAGATAC (p.Gly296_Ser300delinsGlnIlePro). Protein structure analysis indicated that both variants are likely to be pathogenic. Sequencing of SLC25A3 should be considered in patients with isolated cardiomyopathy, even those without generalized skeletal myopathy or lactic acidosis.

One obstacle to retrovirus-mediated gene therapy for human hematopoietic disorders is the low efficiency of gene transfer into pluripotent hematopoietic stem cells (HSC). We have previously shown a direct correlation between retrovirus receptor mRNA levels in mouse HSC and the efficiency with which they are transduced. In the present study, we assayed retrovirus receptor mRNA levels in a variety of mouse and human HSC populations to identify HSC which may be more competent for retrovirus transduction. The highest levels of amphotropic retrovirus receptor (amphoR) mRNA were found in cryopreserved human cord blood HSC. The level of amphoR mRNA in Lin−CD34+ CD38− cells isolated from frozen cord blood was 12-fold higher than the level in fresh cord blood Lin− CD34+ CD38− cells. In mice, the level of amphoR mRNA in HSC from the bone marrow (BM) of mice treated with stem cell factor and granulocyte-colony stimulating factor was 2.8- to 7.8-fold higher than in HSC from the BM of untreated mice. These findings suggest that HSC from frozen cord blood and cytokine-mobilized BM may be superior targets for amphotropic retrovirus transduction compared with HSC from untreated adult BM.

The protection against coronary artery disease attributed to high density lipoprotein (HDL) may be associated with several functions, including its central role in reverse cholesterol transport, possible antioxidant and antithrombotic properties and others not yet identified which may depend on specific interactions between HDL and cell receptors. Several HDL-binding proteins have been identified including two candidate liver HDL receptors, HB1 and HB2 recently purified in this laboratory. We now report the cloning, sequencing, and some properties of HB2, the most abundant of the pair. It shows significant homology with the adhesion molecules ALCAM and BEN of the immunoglobulin superfamily and the cDNA, when transfected into HepG2 or COS cells, caused specific HDL3 binding to increase by 80–100%. Further, ligand blotting of glycoproteins isolated from phorbol 12-myristate 13-acetate-treated THP-1 cells or from transfected HepG2 and Chinese hamster ovary cells also provided evidence of increased binding of HDL3 to HB2. Differentiation of THP-1 cells into macrophages resulted in a striking increase in HB2 mRNA which was attenuated if cells were cholesterol-loaded by incubation with acetylated low density lipoprotein. If the interaction between HDL and HB2 reduces the adhesion-induced inflammatory cellular events that characterize arterial wall injury, thereby achieving the protection associated with higher plasma levels of HDL, these findings may provide a clue to one mitigating effect of HDL in heart disease.

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UCCF-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UCCF-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o− and Fischer rat thyroid cells, expressing various CFTR forms. UCCF-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UCCF-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.

Therapies to correct the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. The β₂ adrenergic receptor agonists, which provide the CFTR stimuli commonly used in nasal potential difference assays, may not overcome the channel gating defects seen in ΔF508 CFTR after plasma membrane localization. In this study, we identify an agent, quercetin, that enhances the detection of surface ΔF508 CFTR, and is suitable for nasal perfusion. A screen of flavonoids in CFBE41o⁻ cells stably transduced with ΔF508 CFTR, corrected to the cell surface with low temperature growth, revealed that quercetin stimulated an increase in the short-circuit current. This increase was dose-dependent in both Fisher rat thyroid and CFBE41o⁻ cells. High concentrations inhibited Cl⁻ conductance. In CFBE41o⁻ airway cells, quercetin (20 μg/ml) activated ΔF508 CFTR, whereas the β₂ adrenergic receptor agonist isoproterenol did not. Quercetin had limited effects on cAMP levels, but did not produce detectable phosphorylation of the isolated CFTR R-domain, suggesting an activation independent of channel phosphorylation. When perfused in the nares of Cftr(+) mice, quercetin (20 μg/ml) produced a hyperpolarization of the potential difference that was absent in Cftr(-/-) mice. Finally, quercetin-induced, dose-dependent hyperpolarization of the nasal potential difference was also seen in normal human subjects. Quercetin activates CFTR-mediated anion transport in respiratory epithelia in vitro and in vivo, and may be useful in studies intended to detect the rescue of ΔF508 CFTR by nasal potential difference.

We examined the activity of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) stably expressed in polarized cystic fibrosis bronchial epithelial cells (CFBE41o(-)) human airway cells and Fisher Rat Thyroid (FRT) cells following treatment with low temperature and a panel of small molecule correctors of DeltaF508 CFTR misprocessing. Corr-4a increased DeltaF508 CFTR-dependent Cl(-) conductance in both cell types, whereas treatment with VRT-325 or VRT-640 increased activity only in FRT cells. Total currents stimulated by forskolin and genistein demonstrated similar dose/response effects to Corr-4a treatment in each cell type. When examining the relative contribution of forskolin and genistein to total stimulated current, CFBE41o(-) cells had smaller forskolin-stimulated I(sc) following either low temperature or corr-4a treatment (10-30% of the total I(sc) produced by the combination of both CFTR agonists). In contrast, forskolin consistently contributed greater than 40% of total I(sc) in DeltaF508 CFTR-expressing FRT cells corrected with low temperature, and corr-4a treatment preferentially enhanced forskolin dependent currents only in FRT cells (60% of total I(sc)). DeltaF508 CFTR cDNA transcript levels, DeltaF508 CFTR C band levels, or cAMP signaling did not account for the reduced forskolin response in CFBE41o(-) cells. Treatment with non-specific inhibitors of phosphodiesterases (papaverine) or phosphatases (endothall) did not restore DeltaF508 CFTR activation by forskolin in CFBE41o(-) cells, indicating that the Cl(-) transport defect in airway cells is distal to cAMP or its metabolism. The results identify important differences in DeltaF508 CFTR activation in polarizing epithelial models of CF, and have important implications regarding detection of rescued of DeltaF508 CFTR in vivo.

In red blood cells ankyrin (ANK-1) provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane. We have previously demonstrated that a 271-bp 5'-flanking region of the ANK-1 gene has promoter activity in erythroid, but not non-erythroid, cell lines. To determine whether the ankyrin promoter could direct erythroid-specific expression in vivo, we analyzed transgenic mice containing the ankyrin promoter fused to the human (A)gamma-globin gene. Sixteen of 17 lines expressed the transgene in erythroid cells indicating nearly position-independent expression. We also observed a significant correlation between the level of Ank/(A)gamma-globin mRNA and transgene copy number. The level of Ank/(A)gamma mRNA averaged 11% of mouse alpha-globin mRNA per gene copy at all developmental stages. The addition of the HS2 enhancer from the beta-globin locus control region to the Ank/(A)gamma-globin transgene resulted in Ank/(A)gamma-globin mRNA expression in embryonic and fetal erythroid cells in six of eight lines but resulted in absent or dramatically reduced levels of Ank/(A)gamma-globin mRNA in adult erythroid cells in eight of eight transgenic lines. These data indicate that the minimal ankyrin promoter contains all sequences necessary and sufficient for erythroid-specific, copy number-dependent, position-independent expression of the human (A)gamma-globin gene.

In order to probe the structural and functional properties of a central region of apolipoprotein A-I (apoA-I), we engineered mutants of the mature form of the protein and expressed them using the baculovirus/insect cell expression system. The mutations which targeted the region of apoA-I between amino acids 140 and 150 included: (i) deletion of the region 140-150 (apoA-I(Delta140-150)); (ii) substitution of arginine 149 with valine (apoA-I(R149V)); (iii) substitution of proline 143 with alanine (apoA-I(P143A)); (iv) deletion of region 63-73 (apoA-I(Delta63-73)), which has structural properties similar to 140-150; and (v) a chimeric protein substituting amino acids 140-150 with amino acids 63-73 (apoA-I(140-150 --> 63-73)). The efficiencies of synthesis were vastly different for the various mutants as follows: apoA-I(R149V) > apoA-I(140-150 --> 63-73) > apoA-I(Delta63-73) > apoA-I(P143A) > apoA-I > apoA-I(Delta140-150). About 50% of the synthesized wild type and all apoA-I mutants was retained in the cells. During expression of apoA-I(R149V) an unusual spontaneous recombination occurred. In addition to the expected mutant, another form of apoA-I with an apparent M(r) of 36K was produced which consisted of a duplication of the amino-terminal end of apoA-I, from the prepeptide through to amino acid 62, linked to the original pre-apoA-I(R149V) sequence via a 4-amino-acid linker. Despite the fact that this form of apoA-I carries two prepeptides and consequently two cleavage sites, there was little, if any, cleavage at the internal cleavage site. During expression, less than 20% of this mutant was retained in the cells. These results demonstrate that at least in the model of insect cells, the efficiency of apoA-I synthesis, processing, and secretion depends on apoA-I secondary structure and/or folding.

ABSTRACT: The low level of amphotropic retrovirus mediated gene transfer into human hematopoietic stem cells (HSC) has been an impediment to gene therapy for hematopoietic diseases (1). We have previously shown that mouse and human HSC have low levels of the mRNA encoding PiT-2, the amphotropic retrovirus receptor. We hypothesized that the low level of PiT-2 mRNA was responsible for the low frequency of transduction of HSC by amphotropic retroviral vectors (2). In this study we compared the level of PiT-2 and PiT-1, the Gibbon Ape Leukemia Virus receptor (GaLV), in 5 human tissue culture cell lines. PiT-2 and PiT-1 mRNA levels were highest in K562 cells and lowest in HL60 cells. In hematopoietic cell lines, the level of PiT-2 or PiT-1 mRNA correlated directly with retrovirus binding and transduction with the appropriate (amphotropic or GaLV) retrovirus vector. The level of expression of PiT-2 and PiT-1 mRNA could be increased by treatment of HL60 cells with either PMA or Interleukin-1α. The increase in the level of PiT-2 and PiT-1 mRNA correlated with increased transduction with both amphotropic and GaLV retroviral vectors. We conclude that the improved transduction was a direct effect of the increased levels of receptor mRNA and unrelated to changes in the cell cycle status.