Unusual context of CENPJ variants and primary microcephaly : compound heterozygosity and nonconsanguinity in an Argentinian patient

Primary microcephaly (MCPH) is a genetically heterogeneous disorder showing an autosomal recessive mode of inheritance. Patients with MCPH present head circumference values two or three standard deviations (SDs) significantly below the mean for age- and sex-matched populations. MCPH is associated with a nonprogressive mild to severe intellectual disability, with normal brain structure in most patients, or with a small brain and gyri without visceral malformations. We present the case of an adult patient born from Argentinian nonconsanguineous healthy parents. He had a head circumference >5 SD below the mean, cerebral neuroimaging showing hypoplasia of the corpus callosum, bilateral migration disorder with heterotopia of the sylvian fissure and colpocephaly. The patient was compound heterozygous for pathogenic variants in the CENPJ gene (c.289dupA inherited from his mother and c.1132 C > T inherited from his father). Our patient represents an uncommon situation for the usual known context of CENPJ and MCPH, including family origin (Argentinian), pedigree (nonconsanguineous), and genotype (a compound heterozygous case with two variants predicting a truncated protein). Next-generation sequencing studies applied in a broader spectrum of clinical presentations of MCPH syndromes may discover additional similar patients and families

Characterization of Novel StAR (Steroidogenic Acute Regulatory Protein) Mutations Causing Non-Classic Lipoid Adrenal Hyperplasia

Context Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH). Objective StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported. Design To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature. Setting Collaboration between the University Children's Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d'Hebron, Autonomous University, Barcelona, Spain. Patients Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age. Results StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (~30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol. Conclusions StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed

Characterization of Novel StAR (Steroidogenic Acute Regulatory Protein) Mutations Causing Non-Classic Lipoid Adrenal Hyperplasia

Context Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH). Objective StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported. Design To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature. Setting Collaboration between the University Children's Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d'Hebron, Autonomous University, Barcelona, Spain. Patients Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age. Results StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (~30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol. Conclusions StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed

Novel variant in HHAT as a cause of different sex development with partial gonadal dysgenesis associated with microcephaly, eye defects, and distal phalangeal hypoplasia of both thumbs: Case report

Different sexual development; Minigene studiesDesarrollo sexual diferente; Estudios de minigenesDesenvolupament sexual diferent; Estudis minigènicsThe palmitoylation of the Hedgehog (Hh) family of morphogens, named sonic hedgehog (SHH), desert hedgehog (DHH), and Indian hedgehog (IHH), is crucial for effective short- and long-range signaling. The hedgehog acyltransferase (HHAT) attaches the palmitate molecule to the Hh; therefore, variants in HHAT cause a broad spectrum of phenotypes. A missense HHAT novel variant c.1001T>A/p.(Met334Lys) was described in a patient first referred for a 46,XY different sexual development with partial gonadal dysgenesis but with microcephaly, eye defects, and distal phalangeal hypoplasia of both thumbs. The in silico analysis of the variant predicted an affectation of the nearest splicing site. Thus, in vitro minigene studies were carried out, which demonstrated that the variant does not affect the splicing. Subsequent protein in silico studies supported the pathogenicity of the variant, and, in conclusion, this was considered the cause of the patient’s phenotype.This study was partly supported by a grant from the Fondo de Investigación Sanitaria (PI15/01647 [to MF-C and SB-S])

Human growth hormone (GH1) gene polymorphism map in a normal-statured adult population

OBJECTIVE: GH1 gene presents a complex map of single nucleotide polymorphisms (SNPs) in the entire promoter, coding and noncoding regions. The aim of the study was to establish the complete map of GH1 gene SNPs in our control normal population and to analyse its association with adult height. DESIGN, SUBJECTS AND MEASUREMENTS: A systematic GH1 gene analysis was designed in a control population of 307 adults of both sexes with height normally distributed within normal range for the same population: −2 standard deviation scores (SDS) to +2 SDS. An analysis was performed on individual and combined genotype associations with adult height. RESULTS: Twenty-five SNPs presented a frequency over 1%: 11 in the promoter (P1 to P11), three in the 5′UTR region (P12 to P14), one in exon 1 (P15), three in intron 1 (P16 to P18), two in intron 2 (P19 and P20), two in exon 4 (P21 and P22) and three in intron 4 (P23 to P25). Twenty-nine additional changes with frequencies under 1% were found in 29 subjects. P8, P19, P20 and P25 had not been previously described. P6, P12, P17 and P25 accounted for 6·2% of the variation in adult height (P = 0·0007) in this population with genotypes A/G at P6, G/G at P6 and A/G at P12 decreasing height SDS (−0·063 ± 0·031, −0·693 ± 0·350 and −0·489 ± 0·265, Mean ± SE) and genotypes A/T at P17 and T/G at P25 increasing height SDS (+1·094 ± 0·456 and +1·184 ± 0·432). CONCLUSIONS: This study established the GH1 gene sequence variation map in a normal adult height control population confirming the high density of SNPs in a relatively small gene. Our study shows that the more frequent SNPs did not significantly contribute to height determination, while only one promoter and two intronic SNPs contributed significantly to it. Studies in larger populations will have to confirm the associations and in vitro functional studies will elucidate the mechanisms involved. Systematic GH1 gene analysis in patients with growth delay and suspected GH deficiency/insufficiency will clarify whether different SNP frequencies and/or the presence of different sequence changes may be associated with phenotypes in them

Expanding the clinical and genetic spectra of primary immunodeficiency-related disorders with clinical exome sequencing: expected and unexpected findings

Inmunodeficiencias primarias; Secuenciación de próxima generación; Secuenciación clínica del exomaImmunodeficiències primàries; Seqüenciació de propera generació; Seqüenciació clínica d’exomesPrimary immunodeficiencies; Next generation sequencing; Clinical exome sequencingPrimary immunodeficiencies (PIDs) refer to a clinically, immunologically, and genetically heterogeneous group of over 350 disorders affecting development or function of the immune system. The increasing use of next-generation sequencing (NGS) technology has greatly facilitated identification of genetic defects in PID patients in daily clinical practice. Several NGS approaches are available, from the unbiased whole exome sequencing (WES) to specific gene panels. Here, we report on a 3-year experience with clinical exome sequencing (CES) for genetic diagnosis of PIDs. We used the TruSight One sequencing panel, which includes 4,813 disease-associated genes, in 61 unrelated patients (pediatric and adults). The analysis was done in 2 steps: first, we focused on a virtual PID panel and then, we expanded the analysis to the remaining genes. A molecular diagnosis was achieved in 19 (31%) patients: 12 (20%) with mutations in genes included in the virtual PID panel and 7 (11%) with mutations in other genes. These latter cases provided interesting and somewhat unexpected findings that expand the clinical and genetic spectra of PID-related disorders, and are useful to consider in the differential diagnosis. We also discuss 5 patients (8%) with incomplete genotypes or variants of uncertain significance. Finally, we address the limitations of CES exemplified by 7 patients (11%) with negative results on CES who were later diagnosed by other approaches (more specific PID panels, WES, and comparative genomic hybridization array). In summary, the genetic diagnosis rate using CES was 31% (including a description of 12 novel mutations), which rose to 42% after including diagnoses achieved by later use of other techniques. The description of patients with mutations in genes not included in the PID classification illustrates the heterogeneity and complexity of PID-related disorders.This study was funded by Instituto de Salud Carlos III, grants PI14/00405 and PI17/00660, cofinanced by the European Regional Development Fund (ERDF)

A crowdsourcing database for the copy-number variation of the Spanish population

Background: Despite being a very common type of genetic variation, the distribution of copy-number variations (CNVs) in the population is still poorly understood. The knowledge of the genetic variability, especially at the level of the local population, is a critical factor for distinguishing pathogenic from non-pathogenic variation in the discovery of new disease variants. Results: Here, we present the SPAnish Copy Number Alterations Collaborative Server (SPACNACS), which currently contains copy number variation profiles obtained from more than 400 genomes and exomes of unrelated Spanish individuals. By means of a collaborative crowdsourcing effort whole genome and whole exome sequencing data, produced by local genomic projects and for other purposes, is continuously collected. Once checked both, the Spanish ancestry and the lack of kinship with other individuals in the SPACNACS, the CNVs are inferred for these sequences and they are used to populate the database. A web interface allows querying the database with different filters that include ICD10 upper categories. This allows discarding samples from the disease under study and obtaining pseudo-control CNV profiles from the local population. We also show here additional studies on the local impact of CNVs in some phenotypes and on pharmacogenomic variants. SPACNACS can be accessed at: http://csvs.clinbioinfosspa.es/spacnacs/. Conclusion: SPACNACS facilitates disease gene discovery by providing detailed information of the local variability of the population and exemplifies how to reuse genomic data produced for other purposes to build a local reference database

Table_3_A Polygenic Risk Score Based on a Cardioembolic Stroke Multitrait Analysis Improves a Clinical Prediction Model for This Stroke Subtype.DOCX

[Background] Occult atrial fibrillation (AF) is one of the major causes of embolic stroke of undetermined source (ESUS). Knowing the underlying etiology of an ESUS will reduce stroke recurrence and/or unnecessary use of anticoagulants. Understanding cardioembolic strokes (CES), whose main cause is AF, will provide tools to select patients who would benefit from anticoagulants among those with ESUS or AF. We aimed to discover novel loci associated with CES and create a polygenetic risk score (PRS) for a more efficient CES risk stratification.[Methods] Multitrait analysis of GWAS (MTAG) was performed with MEGASTROKE-CES cohort (n = 362,661) and AF cohort (n = 1,030,836). We considered significant variants and replicated those variants with MTAG p-value < 5 × 10−8 influencing both traits (GWAS-pairwise) with a p-value < 0.05 in the original GWAS and in an independent cohort (n = 9,105). The PRS was created with PRSice-2 and evaluated in the independent cohort.[Results] We found and replicated eleven loci associated with CES. Eight were novel loci. Seven of them had been previously associated with AF, namely, CAV1, ESR2, GORAB, IGF1R, NEURL1, WIPF1, and ZEB2. KIAA1755 locus had never been associated with CES/AF, leading its index variant to a missense change (R1045W). The PRS generated has been significantly associated with CES improving discrimination and patient reclassification of a model with age, sex, and hypertension.[Conclusion] The loci found significantly associated with CES in the MTAG, together with the creation of a PRS that improves the predictive clinical models of CES, might help guide future clinical trials of anticoagulant therapy in patients with ESUS or AF.Peer reviewe

Regulación por glucocorticoides y vitamina D de la proliferación y la diferenciación de condrocitos de cartílago epifiseal fetal humano

[spa] El objetivo de este trabajo ha consistido en el estudio de la regulación de la proliferación celular y de la expresión de los genes de interés por glucocorticoides (GCs) y vitamina D (VitD), así como analizar el efecto del IGF-I y de la GH sobre la regulación observada por GCs y/o por VitD. Se ha demostrado que los GCs y la VitD regulan la proliferación celular y la expresión de los genes estudiados. El IGF-I y la GH modulan parte de los efectos de los GCs y de la VitD sobre la proliferación celular y la expresión génica. Concentraciones bajas de Dx (10-9 M) no interfieren en la proliferación de los condrocitos mientras que a partir de 10-7 M la inhiben de forma significativa y concentración dependiente. El Mifepristone (MF), bloqueador antagonista del receptor de GCs, revierte la inhibición de la proliferación celular producida por la Dx 10-6 M. La Dx inhibe la expresión de los genes IGF-I, IGFBP3, GHR, SOX9, COL2A1 y aggrecan mientras que estimula la expresión de IGF-IR y COMP. El MF revierte la inhibición que la Dx produce sobre la expresión de GHR, SOX9, COL2A1 y aggrecan pero no la inhibición de la expresión de IGF-I y de IGFBP3. La síntesis de las proteínas IGF-I e IGFBP3 está inhibida por Dx 10-6 de forma paralela a la inhibición de la expresión génica. La VitD inhibe la proliferación celular de forma significativa y concentración dependiente a partir de 10-10 M y estimula la expresión de los genes del eje GH-IGFs (IGF-I, IGFBP3, IGF-IR y GHR) y COMP mientras que inhibe la expresión de SOX9, COL2A1 y aggrecan. La síntesis de las proteínas IGF-I e IGFBP3 está estimulada por VitD de forma paralela a la estimulación de la expresión génica. La expresión de IGF-II no se regula significativamente ni por GCs ni por VitD, por lo tanto, el IGF-I, y no el IGF-II, es el gen regulado por GCs y VitD en condrocitos de cartílago de crecimiento epifiseal fetal humano. El IGF-I es un potente estimulador de la proliferación celular y estimula la expresión de su propio gen, de IGFBP3, de SOX9 y de COL2A1 y aggrecan mientras que inhibe la expresión de IGF-IR y de COMP. El IGF-I revierte la inhibición de la proliferación celular producida por la Dx y por la VitD. El IGF-I revierte la inhibición que la Dx produce sobre la expresión de COL2A1 y aggrecan pero no la producida sobre la expresión de IGF-I, IGFBP3 y de SOX9. La expresión de IGFBP3 está incrementada cuando se incuban los condrocitos con IGF-I y VitD respecto a la incubación con IGF-I o con VitD solos. El IGF-I no revierte la inhibición que la VitD produce sobre la expresión de SOX9 pero sí la producida sobre la expresión de COL2A1 y aggrecan. La GH no tiene efecto significativo sobre la proliferación celular ni sobre la expresión génica en condrocitos de cartílago de crecimiento epifiseal fetal humano en las condiciones experimentales utilizadas. La presencia de GH en el medio de cultivo que contiene además VitD incrementa la expresión de IGF-I y GHR respecto a las células tratadas con VitD sola. El tratamiento conjunto con Dx, GH y VitD revierte la inhibición que la Dx produce sobre la expresión de IGF-I y GHR. Nuestros resultados sugieren que, en tratamientos crónicos con GCs que provocan un retraso de crecimiento, el mantenimiento de niveles óptimos de VitD puede contribuir a mejorar la respuesta al tratamiento con GH.[eng] Circulating supraphysiologic glucocorticoid (GC) concentrations inhibit skeletal growth. Growth plate cartilage and bone are target tissues for vitamin D (Vit D) and deficiency states during infancy (rickets or VitD insensitivity) provoke growth delay. With the aim of analysing the molecular mechanisms involved in skeletal growth inhibition produced by both GC excess and VitD deficiency, as well as the interaction of VitD with GH to oppose gene expression inhibition produced by high GC concentrations, VitD effects on proliferation and gene expression were studied in chondrocytes from human fetal epiphyseal cartilage. Results: Low-dose Dx (10-9 M) maintained proliferation whereas higher concentrations dose-dependently inhibited it. A similar effect was observed with VitD. IGF-I significantly stimulated proliferation and completely opposed the inhibition produced by Dx or VitD. GH had no significant effect. The addition of GH to Dx (10-6 M) and/or VitD (10-6 M) did not limit the inhibitory effects of Dx and/or VitD. Dx dose-dependently inhibited expression of GH-IGF axis genes (IGF-I, IGFBP-3, GHR), transcription factor and matrix proteins (SOX9, COL2A1, aggrecan), whereas it stimulated expression of IGF-IR and the matrix protein COMP. VitD had an opposite effect, which was also dose-dependent, by stimulating expression of IGF-I, IGFBP-3, IGF-IR, GHR and COMP, whereas it inhibited expression of SOX9, COL2A1 and aggrecan. GH alone had a variable effect, which did not reach statistical significance. VitD opposed IGF-I and GHR expression inhibition produced by Dx whereas it did not limit IGFBP-3 inhibition. The combination of VitD with GH not only completely opposed inhibition by Dx of GHR and IGF-I expression, but stimulated it. Conclusions: VitD up-regulated IGF-I, IGFBP-3, GHR and IGF-IR and COMP gene expression in human foetal epiphyseal chondrocytes. Addition of VitD to GH resulted in the highest increase in IGF-I expression. In the presence of pharmacological GC concentrations, VitD rescued IGF-I & GHR gene expression inhibition, although not IGFBP-3. Addition of VitD to GH resulted in significant stimulation of IGF-I expression, even in the presence of GC at high concentrations

Broad phenotypes in heterozygous NR5A1 46,XY patients with a disorder of sex development: an oligogenic origin?

SF-1/NR5A1 is a transcriptional regulator of adrenal and gonadal development. NR5A1 disease-causing variants cause disorders of sex development (DSD) and adrenal failure, but most affected individuals show a broad DSD/reproductive phenotype only. Most NR5A1 variants show in vitro pathogenic effects, but not when tested in heterozygote state together with wild-type NR5A1 as usually seen in patients. Thus, the genotype-phenotype correlation for NR5A1 variants remains an unsolved question. We analyzed heterozygous 46,XY SF-1/NR5A1 patients by whole exome sequencing and used an algorithm for data analysis based on selected project-specific DSD- and SF-1-related genes. The variants detected were evaluated for their significance in literature, databases and checked in silico using webtools. We identified 19 potentially deleterious variants (one to seven per patient) in 18 genes in four 46,XY DSD subjects carrying heterozygous NR5A1 disease-causing variants. We constructed a scheme of all these hits within the landscape of currently known genes involved in male sex determination and differentiation. Our results suggest that the broad phenotype in these heterozygous NR5A1 46,XY DSD subjects may well be explained by an oligogenic mode of inheritance, in which multiple hits, individually non-deleterious, may contribute to a DSD phenotype unique to each heterozygous SF-1/NR5A1 individual