Analysis of germline variants in pediatric patients diagnosed with desmoid tumors and nuchal-type fibromas.

Desmoid tumor (DT) is a fibroblastic proliferation arising in soft tissue characterized by localized infiltrative growth with an inability to metastasize but with a tendency to recurrence. Nuchal-type fibromas are benign soft tissue lesions that are usually developed in the posterior neck. The development of these neoplasms can be associated with a hereditary cancer predisposition syndrome, mainly familial adenomatous polyposis (FAP) syndrome caused by APC germline mutations. Gardner syndrome is a variant of FAP characterized by the presence of extracolonic manifestations including soft tissue tumors as DTs and nuchal-type fibromas. However, the development of these tumors could be associated with germline alterations in other genes related to colorectal cancer development. The objective of this study was to analyze germline variants in APC, MUTYH, POLD1 and POLE genes in five pediatric patients diagnosed with DTs or nuchal-type fibromas. We identified two pathogenic variants in the APC gene in two different patients diagnosed with nuchal-type fibroma and DTs and two variants of uncertain significance in POLD1 in two patients diagnosed with nuchal-type fibroma. Two patients had family history of colorectal cancer, however, only one of them showed an APC germline pathogenic variant. The analysis of germline variants and genetic counseling is essential for pediatric patients diagnosed with DTs or nuchal-type fibromas and their relatives.

Clinical description, molecular delineation and genotype-phenotype correlation in 340 patients with KBG syndrome: addition of 67 new patients.

BACKGROUND: KBG syndrome is a highly variable neurodevelopmental disorder and clinical diagnostic criteria have changed as new patients have been reported. Both loss-of-function sequence variants and large deletions (copy number variations, CNVs) involving ANKRD11 cause KBG syndrome, but no genotype-phenotype correlation has been reported. METHODS: 67 patients with KBG syndrome were assessed using a custom phenotypical questionnaire. Manifestations present in >50% of the patients and a 'phenotypical score' were used to perform a genotype-phenotype correlation in 340 patients from our cohort and the literature. RESULTS: Neurodevelopmental delay, macrodontia, triangular face, characteristic ears, nose and eyebrows were the most prevalentf (eatures. 82.8% of the patients had at least one of seven main comorbidities: hearing loss and/or otitis media, visual problems, cryptorchidism, cardiopathy, feeding difficulties and/or seizures. Associations found included a higher phenotypical score in patients with sequence variants compared with CNVs and a higher frequency of triangular face (71.1% vs 42.5% in CNVs). Short stature was more frequent in patients with exon 9 variants (62.5% inside vs 27.8% outside exon 9), and the prevalence of intellectual disability/attention deficit hyperactivity disorder/autism spectrum disorder was lower in patients with the c.1903_1907del variant (70.4% vs 89.4% other variants). Presence of macrodontia and comorbidities were associated with larger deletion sizes and hand anomalies with smaller deletions. CONCLUSION: We present a detailed phenotypical description of KBG syndrome in the largest series reported to date of 67 patients, provide evidence of a genotype-phenotype correlation between some KBG features and specific ANKRD11 variants in 340 patients, and propose updated clinical diagnostic criteria based on our findings.

Clinical utility of genetic testing in early-onset kidney disease: seven genes are the main players.

BACKGROUND: Inherited kidney diseases are one of the leading causes of chronic kidney disease (CKD) that manifests before the age of 30 years. Precise clinical diagnosis of early-onset CKD is complicated due to the high phenotypic overlap, but genetic testing is a powerful diagnostic tool. We aimed to develop a genetic testing strategy to maximize the diagnostic yield for patients presenting with early-onset CKD and to determine the prevalence of the main causative genes. METHODS: We performed genetic testing of 460 patients with early-onset CKD of suspected monogenic cause using next-generation sequencing of a custom-designed kidney disease gene panel in addition to targeted screening for c.428dupC MUC1. RESULTS: We achieved a global diagnostic yield of 65% (300/460), which varied depending on the clinical diagnostic group: 77% in cystic kidney diseases, 76% in tubulopathies, 67% in autosomal dominant tubulointerstitial kidney disease, 61% in glomerulopathies and 38% in congenital anomalies of the kidney and urinary tract. Among the 300 genetically diagnosed patients, the clinical diagnosis was confirmed in 77%, a specific diagnosis within a clinical diagnostic group was identified in 15%, and 7% of cases were reclassified. Of the 64 causative genes identified in our cohort, 7 (COL4A3, COL4A4, COL4A5, HNF1B, PKD1, PKD2 and PKHD1) accounted for 66% (198/300) of the genetically diagnosed patients. CONCLUSIONS: Two-thirds of patients with early-onset CKD in this cohort had a genetic cause. Just seven genes were responsible for the majority of diagnoses. Establishing a genetic diagnosis is crucial to define the precise aetiology of CKD, which allows accurate genetic counselling and improved patient management.

Prenatal and foetal autopsy findings in glutaric aciduria type II.

BACKGROUND: Glutaric aciduria type 2 is a rare, lethal disorder that affects metabolism of fatty acids caused by genetic defects in electron transfer (ETF) or in electron transfer flavoprotein dehydrogenase (ETFDH). We aimed to describe the pathological findings of 15 week old foetus, born from a consanguineous couple with 3 previous perinatal deaths. The last son died at 4 days of life and genetic analyses revealed a novel probably pathogenic variant at ETFDH (c.706dupG + c.706dupG) that codifies for a truncated protein (p.Glu236Glyfs*5 + p.Glu236Glyfs*5). CASE: During the gestation, due to the medical familial history, prenatal echography and a chorial biopsy for ETFDH-associated glutaric aciduria analysis were carried out. Sanger sequencing confirmed the presence of the homozygous familial variant in the ETFDH gene. The gestation was terminated and the foetal autopsy performed. Autopsy revealed prominent forehead, flat nasal bridge, malformed ears, intrauterine growth retardation, polycystic kidneys and steatosis in the liver, consistent with the diagnosis of glutaric aciduria type II. The comparison of present cases with the previously reported in the literature confirmed the presence of classical criteria, but also revealed the association with urogenital deformities, not previously stated. CONCLUSIONS: Clinical and foetal findings allowed the characterisation of the novel variant (c.706dupG at ETDFH) as pathogenic. Genotype-phenotype relationship is important when studying rare genetic disorders such as glutaric aciduria type II, as variants are usually family-specific, leading to a difficulty in the characterisation of their pathogenicity.

A kidney-disease gene panel allows a comprehensive genetic diagnosis of cystic and glomerular inherited kidney diseases.

Molecular diagnosis of inherited kidney diseases remains a challenge due to their expanding phenotypic spectra as well as the constantly growing list of disease-causing genes. Here we develop a comprehensive approach for genetic diagnosis of inherited cystic and glomerular nephropathies. Targeted next generation sequencing of 140 genes causative of or associated with cystic or glomerular nephropathies was performed in 421 patients, a validation cohort of 116 patients with previously known mutations, and a diagnostic cohort of 207 patients with suspected inherited cystic disease and 98 patients with glomerular disease. In the validation cohort, a sensitivity of 99% was achieved. In the diagnostic cohort, causative mutations were found in 78% of patients with cystic disease and 62% of patients with glomerular disease, mostly familial cases, including copy number variants. Results depict the distribution of different cystic and glomerular inherited diseases showing the most likely diagnosis according to perinatal, pediatric and adult disease onset. Of all the genetically diagnosed patients, 15% were referred with an unspecified clinical diagnosis and in 2% genetic testing changed the clinical diagnosis. Therefore, in 17% of cases our genetic analysis was crucial to establish the correct diagnosis. Complex inheritance patterns in autosomal dominant polycystic kidney disease and Alport syndrome were suspected in seven and six patients, respectively. Thus, our kidney-disease gene panel is a comprehensive, noninvasive, and cost-effective tool for genetic diagnosis of cystic and glomerular inherited kidney diseases. This allows etiologic diagnosis in three-quarters of patients and is especially valuable in patients with unspecific or atypical phenotypes.

Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing.

BACKGROUND: Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS). METHODS: Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies). RESULTS: We identified causative mutations in 17 out of the 40 patients (43%). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes. CONCLUSIONS: Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.

Interaction between a Domain of the Negative Regulator of the Ras-ERK Pathway, SPRED1 Protein, and the GTPase-activating Protein-related Domain of Neurofibromin Is Implicated in Legius Syndrome and Neurofibromatosis Type 1.

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120(GAP). Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.

High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype-Phenotype Correlation.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients.

Exome sequencing identifies CTSK mutations in patients originally diagnosed as intermediate osteopetrosis.

Autosomal Recessive Osteopetrosis is a genetic disorder characterized by increased bone density due to lack of resorption by the osteoclasts. Genetic studies have widely unraveled the molecular basis of the most severe forms, while cases of intermediate severity are more difficult to characterize, probably because of a large heterogeneity. Here, we describe the use of exome sequencing in the molecular diagnosis of 2 siblings initially thought to be affected by "intermediate osteopetrosis", which identified a homozygous mutation in the CTSK gene. Prompted by this finding, we tested by Sanger sequencing 25 additional patients addressed to us for recessive osteopetrosis and found CTSK mutations in 4 of them. In retrospect, their clinical and radiographic features were found to be compatible with, but not typical for, Pycnodysostosis. We sought to identify modifier genes that might have played a role in the clinical manifestation of the disease in these patients, but our results were not informative. In conclusion, we underline the difficulties of differential diagnosis in some patients whose clinical appearance does not fit the classical malignant or benign picture and recommend that CTSK gene be included in the molecular diagnosis of high bone density conditions.

A novel mutation in the IRF6 gene associated with facial asymmetry in a family affected with Van der Woude syndrome.

This report describes a novel missense mutation in the interferon regulation factor 6 (IRF6) gene associated to facial asymmetry. This new feature widens the phenotype spectrum of Van der Woude syndrome (VWS).

El síndrome CHARGE / CHARGE syndrome

El síndrome CHARGE debe su nombre a un acrónimo definido por varios defectos congénitos: coloboma, cardiopatía (heart), atresia de coanas, retraso psicomotor y del crecimiento, genitales anómalos, malformaciones auriculares (ear) y/o sordera. Su causa más frecuente es una mutación del gen de la proteína de unión al cromodominio de la ADN helicasa (CHD7) identificada hasta en un 60 por ciento de los casos y su frecuencia se establece en torno a 1/10.000 nacidos vivos. Presentamos 3 pacientes no relacionados, identificados clínicamente y con distinta mutación en el gen CHD7.

Análisis de la mortalidad neonatal precoz en el Instituto Nacional de Perinatología (1999-2001) / Early neonatal mortality analysis at the Instituto Nacional de Perinatología, 1999-2001

Objetivo: Analizar la mortalidad neonatal precoz en el Instituto Nacional de Perinatología de acuerdo con características perinatales y tiempo de vida. Material y métodos: Análisis de casos en donde se estudiaron las variables: peso, edad gestacional al nacer, causa de muerte y tiempo de vida. Se reportan frecuencias, tasas de mortalidad, tasa de incidencia (por 1,000 nacidos vivos), probabilidad de supervivencia y tablas de sobrevida. Resultados: La tasa de mortalidad fue de 17.13 y la tasa de incidencia de 2.48 muertes/día. Las principales causas de muerte fueron: defectos al nacimiento (34%) e inmadurez (27.8%). De acuerdo con el tiempo de vida, 34.4% de las muertes ocurrieron de 1-59 minutos, 32.4% de 1-23 horas y 33.2% de 1-7 días. Se reporta una relación inversa entre las tasas con el peso y la edad gestacional. La probabilidad de supervivencia fue menor en los menores de 1,000 g y en los menores de 25 semanas de gestación. Conclusiones: La menor mortalidad se reportó en neonatos mayores de 750 g y 26 semanas. Las principales causas de muerte fueron los defectos al nacimiento y la inmadurez.

Objective: The early neonatal mortality was analyzed based on perinatal characteristics and time of neonatal death at the Insituto Nacional de Perinatología. Material and methods: To analyze the cases, we considered birth weight, gestational age, primary causes of death, and time of neonatal death. Rate of neonatal mortality and incidence (per 1,000 live births) and survival tables were reported. Results: The neonatal mortality rate was 17.13; the incidence was 2.48 deaths/day. Congenital birth defects (34%) and immaturity (27%) were the principal causes of death. According to the time of death, 34.4% of the deaths happened in the first 59 minutes, 32.4% from one to 23 hours, and 33.2% from one to seven days. There was an inverse relationship between rates, related with birth weight and gestational age. The probability of survival was less in neonates with a weight less than 1,000 g and below 25 weeks. Conclusions: Those neonates whose birth weight was above 750 g and gestational age above 26 weeks had a low mortality rate. The main causes of death were congenital birth defects and immaturity.

Microtia: A clinical and genetic study at the National Institute of Pediatrics in Mexico City

Background. Microtia is a malformation of the ear with extreme variability of expression. It is generally seean as an isolated malformation. However, some authors consider it to be a minimal manifestation of the oculo-auriculo-vertebral spectrum (OAVS), wherem, in addition, there are facial, vertebral, and renal abnormalities, among others. Methods. A total of 145 pediatric patients with unilateral or bilateral microtia not considered as part of a syndrome were studied. All patients were subjected to an international clinical examination, a familial history, and radiographic imaging studies for ruling out associated malformations. Patients were classified into two groups: group 1 (60 percent), with isolated microtia; and group 2 (40 percent), considered as OAVS, with microtia associated with hemifacial skeletal microsomia, vertebral and/or renal malformations. Results. No significant differences were found between the groups when the following variables were compared: gender; prescence of unilateral or bilateral microtia; atretic external auditory canal; presence of preauricelar tags; hearing loss of any type, and affection of the seventh cranial nerve, as well as associated malformations of other otgans or systems. There were significant differences in relation to the presence of soft-tissue hemifacial microsomia, more frequently seen in patients with OAVS, because the majority of these patients had bone microsomia. Over 66 percent of the cases were sporadic and the rest were familiar. In 28.3 percent of the cases, the history suggested an autosomal-dominant inheritance pattern, and in 5.5 percent, an autosomal-recessive inheritance pattent, although in some familial cases, multifactorial inheritance could not be ignored . Some members in several familes has isolated microtia, and others had mild characteristic manifestations of OAVS. Conclusions. Our results support the hypothesis that isolated microtia is a minimal expression of OAVS. Therefore, it is recommended that patients with microtia be subjected to intentional studies that search for malformations and physical examinations of firstdegree relatives for adquate genetic counseling and management