Temporal patterns of osteoclast formation and activity following withdrawal of RANKL inhibition.

Rebound bone loss following denosumab discontinuation is an important clinical challenge. Current treatment strategies to prevent this fail to suppress the rise and overshoot in osteoclast-mediated bone resorption. In this study, we use a murine model of denosumab treatment and discontinuation to show the temporal changes in osteoclast formation and activity during RANKL inhibition and withdrawal. We show that the cellular processes that drive the formation of osteoclasts and subsequent bone resorption following withdrawal of RANKL inhibition precede the rebound bone loss. Furthermore, a rise in serum TRAP and RANKL levels is detected before markers of bone turnover used in current clinical practice. These mechanistic advances may provide insight into a more defined window of opportunity to intervene with sequential therapy following denosumab discontinuation.

Stopping denosumab, a medication commonly used to improve bone mass by blocking formation of bone resorbing osteoclasts, leads to a rebound loss in the bone which was gained during treatment. Current strategies to prevent this bone loss fail in most cases as they are unable to prevent the rise and overshoot in bone resorption by osteoclasts. Thie stems from an incomplete understanding of how osteoclasts behave during denosumab treatment and after treatment is discontinued. We use a mouse model of this phenomenon to show how osteoclast formation and activity changes throughout this process. We show that increases in the processes that drive the formation of osteoclasts can be detected in the circulation before bone loss occurs. These findings could therefore provide insight into a targeted 'window of opportunity' to intervene and prevent the rebound bone loss following stopping denosumab in patients.

Machine Learning Prediction of ADHD Severity: Association and Linkage to ADGRL3, DRD4, and SNAP25.

OBJECTIVE: To investigate whether single nucleotide polymorphisms (SNPs) in the ADGRL3, DRD4, and SNAP25 genes are associated with and predict ADHD severity in families from a Caribbean community. METHOD: ADHD severity was derived using latent class cluster analysis of DSM-IV symptomatology. Family-based association tests were conducted to detect associations between SNPs and ADHD severity latent phenotypes. Machine learning algorithms were used to build predictive models of ADHD severity based on demographic and genetic data. RESULTS: Individuals with ADHD exhibited two seemingly independent latent class severity configurations. SNPs harbored in DRD4, SNAP25, and ADGRL3 showed evidence of linkage and association to symptoms severity and a potential pleiotropic effect on distinct domains of ADHD severity. Predictive models discriminate severe from non-severe ADHD in specific symptom domains. CONCLUSION: This study supports the role of DRD4, SNAP25, and ADGRL3 genes in outlining ADHD severity, and a new prediction framework with potential clinical use.

¿Psicoanálisis y desarrollo? Aportes desde una perspectiva no lineal / Psychoanalysis and Development? Contributions from a non-Linear Perspective

Resumen El presente artículo problematiza la relación entre psicoanálisis freudiano y la perspectiva del desarrollo lineal, determinista y teleológica. A pesar de las impregnaciones evolucionistas del contexto histórico en que Freud gesta sus ideas, se encuentran conceptos que muestran una fuerte ruptura con la idea de un despliegue guiado por el ritmo de etapas prefijadas por edades. Así se postula la a-temporalidad de los procesos inconscientes y la Nachträglichkeit a la luz de las perspectivas no lineales emergentes desde las ciencias de la complejidad. Finalmente, se reflexiona sobre una psique que, en su carácter abierto y complejo, contempla lo aleatorio, lo impredecible y el azar en su devenir, y se transforma mediante trabajo elaborativo simbólico del yo. Así, sólo la historización, nunca lineal, contempla lo nuevo como posibilidad de (re)constituir la historia pasada.

Abstract This article problematizes the relationship between Freudian psychoanalysis and the linear, deterministic, and teleological perspective on development. Despite the evolutionist impregnations of the historical context in which Freud conceives his ideas, there are concepts that show a sharp break with the idea of an unfolding guided by a rhythm of stages prefixed by ages. Thus, the a-temporality of the unconscious processes and the Nachträglichkeit is postulated considering the non-linear perspectives that emerge from the sciences of complexity. Finally, we reflect on a psyche that, in its open and complex character, contemplates the fortuitous, the unpredictable and chance in its becoming, and is transformed through the elaborative symbolic work on the self. Like this, only the historicization, which is never linear, contemplates what is new as a possibility to (re)construct the past history.

Exome Sequencing and Congenital Heart Disease in Sub-Saharan Africa.

BACKGROUND: Congenital heart disease (CHD) is the most common birth defect and affects roughly 1% of the global population. There have been many large CHD sequencing projects in developing countries but none in sub-Saharan Africa. In this exome sequencing study, we recruited families from Lagos, Nigeria, affected by structural heart disease. METHODS: Ninety-eight participants with CHD and an average age of 3.6 years were recruited from Lagos, Nigeria. Exome sequencing was performed on probands and parents when available. For genes of high interest, we conducted functional studies in Drosophila using a cardiac-specific RNA interference-based gene silencing system. RESULTS: The 3 most common CHDs were tetralogy of Fallot (20%), isolated ventricular septal defect (14%), and transposition of the great arteries (8%). Ten percent of the cohort had pathogenic or likely pathogenic variants in genes known to cause CHD. In 64 complete trios, we found 34 de novo variants that were not present in the African population in the Genome Aggregation Database (v3). Nineteen loss of function variants were identified using the genome-wide distribution of selection effects for heterozygous protein-truncating variants (shet). Nine genes caused a significant mortality when silenced in the Drosophila heart, including 4 novel disease genes not previously associated with CHD (UBB, EIF4G3, SREBF1, and METTL23). CONCLUSIONS: This study identifies novel candidate genes and variants for CHD and facilitates comparisons with previous CHD sequencing studies in predominantly European cohorts. The study represents an important first step in genomic studies of CHD in understudied populations. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01952171.

Prevalence of Dirofilaria immitis in shelter dogs in Bucaramanga metropolitan area, Colombia.

The heartworm Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariosis in dogs and cats and also infects humans. The current knowledge about this parasite in South America is scarce compared to North America and Europe. Nevertheless, this parasite has been reported in some regions of Colombia. The objective of this study was to determine the prevalence and risk factors associated with D. immitis infection in shelter dogs from Bucaramanga metropolitan area. We performed a cross-sectional study using blood samples from shelter dogs. Microfilariae testing of whole blood included a blood smear and modified Knott's test. Antigen testing of serum was performed using the Anigen Rapid HW Ag Test Kit 2.0 ®. A descriptive analysis and univariable logistic regression analysis were applied. A total of 207 whole blood samples, from 7 shelters dogs in the Bucaramanga metropolitan area, were utilized. The overall prevalence of D. immitis observed in shelter dogs in the Bucaramanga metropolitan area by blood smears and modified Knott's test was 6.3% (13/ 207) and 0.5% (1/207) by immunochromatography test kit. Regarding the prevalence by municipalities, there was a statistical association (P < .05), indicating that the prevalence was higher in dogs in Girón municipality. Likewise, there was a statistical association (P < .05) between the prevalence with respect to heartworm knowledge and mosquito control. For prevalence by gender, age group, dog breed, and hair length, there was no statistical association (P > .05). For Girón municipality there was a 7.1 times (OR = 7.1; CI95% 0.8-59.2; P < .05) increased infection risk. This study provides current data and the first known report of the prevalence of D. immitis microfilaremia and antigenemia in shelter dogs in the Bucaramanga metropolitan area, and these results could be useful for designing new control measures for this infection. We observed that the prevalence of both microfilaremia and antigenemia was significantly lower in these sampled dogs compared to previous reports.

Determination of organochlorine and organophosphorus residues in surface waters from the coffee zone in Quindío, Colombia.

The aim of this study was to identify organochlorine (OC) and organophosphorus (OP) pesticides levels in water samples collected in secondary water bodies in agricultural area planted with coffee and plantain. A descriptive cross-sectional study was carried out. A validated method for microwave-assisted extraction and gas chromatography with electron microcapture detector (MAE-GC-µECD) was used to analyze pesticide residues in samples. The determinations were based on certified reference material, Organochlorine Pesticide Mix AB #3, Canadian Drinking Organophosphorus Pesticides Mix, and pentachloronitrobenzene (ISTD) Internal Standard Mix 508.1. Pesticide residues were found in 81.3% of the samples, including OCs: 4.4'-DDT (38%), endosulfan II (19.7%), endosulfan sulfate, and endrin (11.7% and 8.8%), and others identified as 4.4'-DDE, Delta-HCB, parathion, chlorpyrifos, endrin aldehyde, heptachlor, heptachlor epoxide, endrin ketone, and methoxychlor. Parathion and/or chlorpyrifos were found in 5.8-8% of samples; the water bodies most heavily affected were those in Filandia and Quimbaya in which 100% of samples were contaminated, followed by those in Calarcá, Córdoba, Pijao, and Génova, with contamination found in over 75% of samples. The results indicated that surface waters from Quindío municipalities are contaminated with pesticide residues hazardous to human health, which are still in use despite being either restricted or prohibited.

Mutations in sphingolipid metabolism genes are associated with ADHD.

Attention deficit hyperactivity disorder (ADHD) is the most prevalent neurodevelopmental disorder in children, with genetic factors accounting for 75-80% of the phenotypic variance. Recent studies have suggested that ADHD patients might present with atypical central myelination that can persist into adulthood. Given the essential role of sphingolipids in myelin formation and maintenance, we explored genetic variation in sphingolipid metabolism genes for association with ADHD risk. Whole-exome genotyping was performed in three independent cohorts from disparate regions of the world, for a total of 1520 genotyped subjects. Cohort 1 (MTA (Multimodal Treatment study of children with ADHD) sample, 371 subjects) was analyzed as the discovery cohort, while cohorts 2 (Paisa sample, 298 subjects) and 3 (US sample, 851 subjects) were used for replication. A set of 58 genes was manually curated based on their roles in sphingolipid metabolism. A targeted exploration for association between ADHD and 137 markers encoding for common and rare potentially functional allelic variants in this set of genes was performed in the screening cohort. Single- and multi-locus additive, dominant and recessive linear mixed-effect models were used. During discovery, we found statistically significant associations between ADHD and variants in eight genes (GALC, CERS6, SMPD1, SMPDL3B, CERS2, FADS3, ELOVL5, and CERK). Successful local replication for associations with variants in GALC, SMPD1, and CERS6 was demonstrated in both replication cohorts. Variants rs35785620, rs143078230, rs398607, and rs1805078, associated with ADHD in the discovery or replication cohorts, correspond to missense mutations with predicted deleterious effects. Expression quantitative trait loci analysis revealed an association between rs398607 and increased GALC expression in the cerebellum.

Generation of human induced pluripotent stem cell line (NIDCRi001-A) from a Muenke syndrome patient with an FGFR3 p.Pro250Arg mutation.

Muenke syndrome is the leading genetic cause of craniosynostosis and results in a variety of disabling clinical phenotypes. To model the disease and study the pathogenic mechanisms, a human induced pluripotent stem cell (hiPSC) line was generated from a patient diagnosed with Muenke syndrome. Successful reprogramming was validated by morphological features, karyotyping, loss of reprogramming factors, expression of pluripotency markers, mutation analysis and teratoma formation.

Biallelic variants in KYNU cause a multisystemic syndrome with hand hyperphalangism.

Catel-Manzke syndrome is characterized by the combination of Pierre Robin sequence and radial deviation, shortening as well as clinodactyly of the index fingers, due to an accessory ossification center. Mutations in TGDS have been identified as one cause of Catel-Manzke syndrome, but cannot be found as causative in every patient with the clinical diagnosis. We performed a chromosome microarray and/or exome sequencing in three patients with hand hyperphalangism, heart defect, short stature, and mild to severe developmental delay, all of whom were initially given a clinical diagnosis of Catel-Manzke syndrome. In one patient, we detected a large deletion of exons 1-8 and the missense variant c.1282C > T (p.Arg428Trp) in KYNU (NM_003937.2), whereas homozygous missense variants in KYNU were found in the other two patients (c.989G > A (p.Arg330Gln) and c.326G > C (p.Trp109Ser)). Plasma and urine metabolomic analysis of two patients indicated a block along the tryptophan catabolic pathway and urine organic acid analysis showed excretion of xanthurenic acid. Biallelic loss-of-function mutations in KYNU were recently described as a cause of NAD deficiency with vertebral, cardiac, renal and limb defects; however, no hand hyperphalangism was described in those patients, and Catel-Manzke syndrome was not discussed as a differential diagnosis. In conclusion, we present unrelated patients identified with biallelic variants in KYNU leading to kynureninase deficiency and xanthurenic aciduria as a very likely cause of their hyperphalangism, heart defect, short stature, and developmental delay. We suggest performance of urine organic acid analysis in patients with suspected Catel-Manzke syndrome, particularly in those with cardiac or vertebral defects or without mutations in TGDS.

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations.

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

Correction: The CHD4-related syndrome: a comprehensive investigation of the clinical spectrum, genotype-phenotype correlations, and molecular basis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of a novel PCNT founder pathogenic variant in the Israeli Druze population.

Majewski Osteodysplastic Primordial Dwarfism type II (MOPDII) is a form of dwarfism associated with severe microcephaly, characteristic skeletal findings, distinct dysmorphic features and increased risk for cerebral infarctions. The condition is caused by bi-allelic loss-of-function variants in the gene PCNT. Here we describe the identification of a novel founder pathogenic variant c.3465-1G > A observed in carriers from multiple Druze villages in Northern Israel. RNA studies show that the variant results in activation of a cryptic splice site causing a coding frameshift. The study was triggered by the diagnosis of a single child with MOPDII and emphasizes the advantages of applying next generation sequencing technologies in community genetics and the importance of establishing population-specific sequencing databases.

The CHD4-related syndrome: a comprehensive investigation of the clinical spectrum, genotype-phenotype correlations, and molecular basis.

PURPOSE: Sifrim-Hitz-Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants inCHD4. In this study, we investigated the clinical spectrum of the disorder, genotype-phenotype correlations, and the effect of different missense variants on CHD4 function. METHODS: We collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains. RESULTS: The majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype-phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains. CONCLUSION: The CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.

Cohesin complex-associated holoprosencephaly.

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.

Novel heterozygous variants in KMT2D associated with holoprosencephaly.

Lysine methyltransferase 2D (KMT2D; OMIM 602113) encodes a histone methyltransferase involved in transcriptional regulation of the beta-globin and estrogen receptor as part of a large protein complex known as activating signal cointegrator-2-containing complex (ASCOM). Heterozygous germline mutations in the KMT2D gene are known to cause Kabuki syndrome (OMIM 147920), a developmental multisystem disorder. Neither holoprosencephaly nor other defects in human forebrain development have been previously associated with Kabuki syndrome. Here we report two patients diagnosed with alobar holoprosencephaly in their antenatal period with de novo monoallelic KMT2D variants identified by trio-based exome sequencing. The first patient was found to have a stop-gain variant c.12565G>T (p.Gly4189*), while the second patient had a missense variant c.5A>G (p.Asp2Gly). Phenotyping of each patient did not reveal any age-related feature of Kabuki syndrome. These two cases represent the first report on association between KMT2D and holoprosencephaly.

A CCR4-NOT Transcription Complex, Subunit 1, CNOT1, Variant Associated with Holoprosencephaly.

Holoprosencephaly is the incomplete separation of the forebrain during embryogenesis. Both genetic and environmental etiologies have been determined for holoprosencephaly; however, a genetic etiology is not found in most cases. In this report, we present two unrelated individuals with semilobar holoprosencephaly who have the identical de novo missense variant in the gene CCR4-NOT transcription complex, subunit 1 (CNOT1). The variant (c.1603C>T [p.Arg535Cys]) is predicted to be deleterious and is not present in public databases. CNOT1 has not been previously associated with holoprosencephaly or other brain malformations. In situ hybridization analyses of mouse embryos show that Cnot1 is expressed in the prosencephalic neural folds at gestational day 8.25 during the critical period for subsequent forebrain division. Combining human and mouse data, we show that CNOT1 is associated with incomplete forebrain division.

ADGRL3 (LPHN3) variants predict substance use disorder.

Genetic factors are strongly implicated in the susceptibility to develop externalizing syndromes such as attention-deficit/hyperactivity disorder (ADHD), oppositional defiant disorder, conduct disorder, and substance use disorder (SUD). Variants in the ADGRL3 (LPHN3) gene predispose to ADHD and predict ADHD severity, disruptive behaviors comorbidity, long-term outcome, and response to treatment. In this study, we investigated whether variants within ADGRL3 are associated with SUD, a disorder that is frequently co-morbid with ADHD. Using family-based, case-control, and longitudinal samples from disparate regions of the world (n = 2698), recruited either for clinical, genetic epidemiological or pharmacogenomic studies of ADHD, we assembled recursive-partitioning frameworks (classification tree analyses) with clinical, demographic, and ADGRL3 genetic information to predict SUD susceptibility. Our results indicate that SUD can be efficiently and robustly predicted in ADHD participants. The genetic models used remained highly efficient in predicting SUD in a large sample of individuals with severe SUD from a psychiatric institution that were not ascertained on the basis of ADHD diagnosis, thus identifying ADGRL3 as a risk gene for SUD. Recursive-partitioning analyses revealed that rs4860437 was the predominant predictive variant. This new methodological approach offers novel insights into higher order predictive interactions and offers a unique opportunity for translational application in the clinical assessment of patients at high risk for SUD.

Low-level parental mosaicism affects the recurrence risk of holoprosencephaly.

PURPOSE: De novo variants (DNVs) represent an important fraction of the pathogenic variant burden in holoprosencephaly (HPE). However, unexpected recurrences can occur, as evidenced by multiple affected children harboring the same apparently DNV. This study was performed to estimate the rate of parental mosaicism in a cohort of patients with HPE. METHODS: We developed a targeted capture next-generation sequencing (NGS) panel of 153 genes with potential implication in HPE. Sequencing data from a cohort of 136 HPE family trios were analyzed to identify probands with apparently DNVs. DNVs were examined in the proband and their parents to detect any deviations from the expected ~50/50 allele ratio of true heterozygosity. Selected variants were confirmed by Droplet Digital™ polymerase chain reaction (ddPCR). RESULTS: We identified 28 high-confidence DNVs, 20 of which occurred in known HPE genes. Nineteen of the 20 variants (95%) were pathogenic or likely pathogenic. Sequence data analysis showed evidence of parental mosaicism in five cases, for an overall mosaicism rate of 26%. In addition, we found evidence for likely postzygotic events in four cases (50%). CONCLUSIONS: High sensitivity methods, such as high-depth NGS and ddPCR, are essential to providing an accurate assessment of recurrence risk in HPE families with apparently DNVs.