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])

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)

Molecular charaterization of Primary Ciliary Dyskinesia

Introduction: Motile cilia are highly complex hair-like organelles protruding from the apical surface of epithelia cells of various human organ systems. They contain a 9+2 tubulin-based axoneme core structure and an important number of multiprotein complexes comprising the sub-structures: the dynein arms, the nexin links, the central sheath and the radial spokes. Primary ciliary dyskinesia (PCD) is an autosomal recessive rare disease (1-15,000 live-born children) caused by an alteration of ciliary structure and function, which impairs the clearance of respiratory secretions. Its clinical manifestations include chronic wet cough, secretory otitis media, rhinosinusitis, repeated episodes of bronchitis and/or recurrent pneumonia, bronchiectasis, male infertility, female subfertility, and situs inversus (50%) or heterotaxia (6%). PCD diagnosis is complex and based on a combination of techniques. Genetics and immunofluorescence have been recently proposed as reliable techniques to improve understanding of disease-causing genes and diagnosis rate in PCD. Objectives: Optimize genetic and immunofluorescence analysis to improve the diagnosis rate in our cohort and help understand the correlation between a specific genetic defect and ciliary structure and function. Methods: This was a multicenter cross-sectional study of patients with a high suspicion of PCD according to the European Respiratory Society criteria. To characterize the genetic alterations in our cohort, we designed a gene panel for massive sequencing that included 44 genes associated with PCD. Whole-exome sequencing was conducted on patients with a gene panel negative result. Immunofluorescence studies were carried out applying a four commercial fluorescently labeled antibody panel (DNAH5, DNALI1, GAS8 and RSPH4A or RSPH9) to study the presence and distribution of these ciliary proteins in nasal brushing respiratory cell samples. Results: Seventy-nine patients, 53 of whom had a diagnosis of confirmed or highly-likely PCD, were included in the genetic study. The sensitivity of the 44 PCD gene panel was 81.1%, with a specificity of 100%. Candidate variants were found in some of the genes of the panel in 43 patients with PCD, 51.2% (22/43) of whom were homozygotes and 48.8% (21/43) compound heterozygotes. The most common causative genes were DNAH5 and CCDC39. We found 52 different variants, 36 of which were not previously described in the literature and the most prevalent variant was detected in RSPH1 (c.85G>T/p.Glu39Ter). In nine patients with gene panel negative result and in one patient firstly considered unlikely PCD, whole-exome sequencing was carried out. Two patients presented candidate variants in two recently genes associated with PCD: CFAP300/C11orf70 and DNAAF6/PIH1D3. Four patients presented variants in candidate genes (GOLGA3 and C2CD3) which could explain their phenotype. Regarding immunofluorescence analysis, the four antibody-panel was tested in 74 nasal brushing samples. Sixty-eight (91.9%) patients were evaluable for all tested antibodies. Thirty-three cases (44.6%) presented an absence or mislocation of protein in ciliary axoneme (15 absent and 3 proximal distribution of DNAH5 in the ciliary axoneme, 3 absent DNAH5 and DNALI1, 7 absent DNALI1 and cytoplasmatic localization of GAS8, 1 absent GAS8, 3 absent RSPH9 and 1 absent RSPH4A). Fifteen patients had confirmed or highly-likely PCD but normal immunofluorescence results (68.8% sensitivity and 100% specificity), three of them with confirmed likely pathogenic variants in DNAH11. Considering our immunofluorescence results we proposed a two-step antibody panel: a first round with DNAH5, DNALI1, GAS8 and RSPH9 antibodies, and a second round, if required, with DNAH11 and SPEF2 antibodies. Conclusions: The design and implementation of our gene panel produces a high yield in the genetic diagnosis of PCD. Whole-exome sequencing analysis is a useful technique in PCD suspected cases with non-genetic variants in known PCD causing genes. Immunofluorescence analysis is a quick, available, low-cost and reliable diagnostic test for PCD, although it cannot be used as a standalone test

Immunofluorescence analysis as a diagnostic tool in a spanish cohort of patients with suspected primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is an autosomal recessive rare disease caused by an alteration of ciliary structure. Immunofluorescence, consisting in the detection of the presence and distribution of cilia proteins in human respiratory cells by fluorescence, has been recently proposed as a technique to improve understanding of disease-causing genes and diagnosis rate in PCD. The objective of this study is to determine the accuracy of a panel of four fluorescently labeled antibodies (DNAH5, DNALI1, GAS8 and RSPH4A or RSPH9) as a PCD diagnostic tool in the absence of transmission electron microscopy analysis. The panel was tested in nasal brushing samples of 74 patients with clinical suspicion of PCD. Sixty-eight (91.9%) patients were evaluable for all tested antibodies. Thirty-three cases (44.6%) presented an absence or mislocation of protein in the ciliary axoneme (15 absent and 3 proximal distribution of DNAH5 in the ciliary axoneme, 3 absent DNAH5 and DNALI1, 7 absent DNALI1 and cytoplasmatic localization of GAS8, 1 absent GAS8, 3 absent RSPH9 and 1 absent RSPH4A). Fifteen patients had confirmed or highly likely PCD but normal immunofluorescence results (68.8% sensitivity and 100% specificity). In conclusion, immunofluorescence analysis is a quick, available, low-cost and reliable diagnostic test for PCD, although it cannot be used as a standalone test

Expanding the Clinical and Genetic Spectra of Primary Immunodeficiency-Related Disorders With Clinical Exome Sequencing : Expected and Unexpected Findings

We are deeply grateful to the affected individuals who participated in this study and their families. We thank the Barcelona PID Foundation for patient support and for funding MG-P. We acknowledge Celine Cavallo for English language support. Funding. This study was funded by Instituto de Salud Carlos III, grants PI14/00405 and PI17/00660, cofinanced by the European Regional Development Fund (ERDF).Primary 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

Immunofluorescence Analysis as a Diagnostic Tool in a Spanish Cohort of Patients with Suspected Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is an autosomal recessive rare disease caused by an alteration of ciliary structure. Immunofluorescence, consisting in the detection of the presence and distribution of cilia proteins in human respiratory cells by fluorescence, has been recently proposed as a technique to improve understanding of disease-causing genes and diagnosis rate in PCD. The objective of this study is to determine the accuracy of a panel of four fluorescently labeled antibodies (DNAH5, DNALI1, GAS8 and RSPH4A or RSPH9) as a PCD diagnostic tool in the absence of transmission electron microscopy analysis. The panel was tested in nasal brushing samples of 74 patients with clinical suspicion of PCD. Sixty-eight (91.9%) patients were evaluable for all tested antibodies. Thirty-three cases (44.6%) presented an absence or mislocation of protein in the ciliary axoneme (15 absent and 3 proximal distribution of DNAH5 in the ciliary axoneme, 3 absent DNAH5 and DNALI1, 7 absent DNALI1 and cytoplasmatic localization of GAS8, 1 absent GAS8, 3 absent RSPH9 and 1 absent RSPH4A). Fifteen patients had confirmed or highly likely PCD but normal immunofluorescence results (68.8% sensitivity and 100% specificity). In conclusion, immunofluorescence analysis is a quick, available, low-cost and reliable diagnostic test for PCD, althouh it cannot be used as a standalone tes

Implementation of a Gene Panel for Genetic Diagnosis of Primary Ciliary Dyskinesia

Introduction: Primary ciliary dyskinesia (PCD) is characterized by an alteration in the ciliary structure causing difficulty in the clearance of respiratory secretions. Diagnosis is complex and based on a combination of techniques. The objective of this study was to design a gene panel including all known causative genes, and to corroborate their diagnostic utility in a cohort of Spanish patients. Methods: This was a multicenter cross-sectional study of patients with a high suspicion of PCD, according to European Respiratory Society criteria, designed around a gene panel for massive sequencing using SeqCap EZ capture technology that included 44 genes associated with PCD. Results: We included 79 patients, 53 of whom had a diagnosis of confirmed or highly probable PCD. The sensitivity of the gene panel was 81.1%, with a specificity of 100%. Candidate variants were found in some of the genes of the panel in 43 patients with PCD, 51.2% (22/43) of whom were homozygotes and 48.8% (21/43) compound heterozygotes. The most common causative genes were DNAH5 and CCDC39. We found 52 different variants, 36 of which were not previously described in the literature. Conclusions: The design and implementation of a tailored gene panel produces a high yield in the genetic diagnosis of PCD. This panel provides a better understanding of the causative factors involved in these patients and lays down the groundwork for future therapeutic approaches

Incidence and Prevalence of Children's Diffuse Lung Disease in Spain.

Children's diffuse lung disease, also known as children's Interstitial Lung Diseases (chILD), are a heterogeneous group of rare diseases with relevant morbidity and mortality, which diagnosis and classification are very complex. Epidemiological data are scarce. The aim of this study was to analyse incidence and prevalence of chILD in Spain. Multicentre observational prospective study in patients from 0 to 18 years of age with chILD to analyse its incidence and prevalence in Spain, based on data reported in 2018 and 2019. A total of 381 cases with chILD were notified from 51 paediatric pulmonology units all over Spain, covering the 91.7% of the paediatric population. The average incidence of chILD was 8.18 (CI 95% 6.28-10.48) new cases/million of children per year. The average prevalence of chILD was 46.53 (CI 95% 41.81-51.62) cases/million of children. The age group with the highest prevalence were children under 1 year of age. Different types of disorders were seen in children 2-18 years of age compared with children 0-2 years of age. Most frequent cases were: primary pulmonary interstitial glycogenosis in neonates (17/65), neuroendocrine cell hyperplasia of infancy in infants from 1 to 12 months (44/144), idiopathic pulmonary haemosiderosis in children from 1 to 5 years old (13/74), hypersensitivity pneumonitis in children from 5 to 10 years old (9/51), and scleroderma in older than 10 years old (8/47). We found a higher incidence and prevalence of chILD than previously described probably due to greater understanding and increased clinician awareness of these rare diseases