GENETIC CHARACTERISTICS AND EXTRACOLONIC PHENOTYPIC MANIFESTATIONS IN PATIENTS WITH FAMILIAR ADENOMATOUS POLYPOSIS. / CARACTERÍSTICAS GENÉTICAS Y MANIFESTACIONES FENOTÍPICAS EXTRACOLÓNICAS EN PACIENTES CON POLIPOSIS ADENOMATOSA FAMILIAR.

INTRODUCTION: Familial adenomatous polyposis (FAP) is a hereditary disease caused by mutations in the APC gene, which is also associated with extracolonic manifestations. The objective was to characterize the extracolonic manifestations in a cohort of patients with classic FAP and the possible genotype-phenotype association. MATERIALS AND METHODS: The study design was observational and descriptive. Demographic, clinical, and genetic variables were collected based on the type of mutation (frameshift, nonsense, splicing, rearrangement, and others). RESULTS: We included 45 patients with FAP (mean age 47 years, range 21-78; 51% female), belonging to 21 families, with a median of 2 (range 0-6) manifestations per patient. 80% (n=36) had upper digestive tract involvement, with duodenal adenomas (73%), fundic gland polyposis (56%), and ampullary adenoma (36%) being the most frequent findings. The most common extraintestinal manifestations were desmoid tumors (16%) and papillary thyroid carcinoma (13%). 38% of the patients presented an aggressive phenotype (Spigelman III-IV, high-grade dysplasia, invasive neoplasia, desmoid tumor, and papillary thyroid carcinoma). The most common genetic mutations were frameshift (56%), nonsense (26%), and splicing (16%), primarily located in exon 15 (50%). No significant correlation was found between the type of genetic mutation and the severity or location of phenotypic manifestations. CONCLUSIONS: One-third of patients with FAP present an aggressive phenotype, without a demonstrated correlation between the type of genetic alteration and the phenotypic manifestations.

Hidden etiology of cerebral palsy: genetic and clinical heterogeneity and efficient diagnosis by next-generation sequencing.

Cerebral palsy (CP) is a heterogeneous neurodevelopmental disorder that causes movement and postural disabilities. Recent research studies focused on genetic diagnosis in patients with CP of unknown etiology. The present study was carried out in 20 families with one family member affected with idiopathic CP. Chromosomal microarray and exome sequencing techniques were performed in all patients. Chromosomal microarray analysis did not show any pathological or probable pathological structural variant. However, the next-generation sequencing study showed a high diagnostic yield. We report 11/20 patients (55%) with different pathogenic or potentially pathogenic variants detected by exome sequencing analysis: five patients with mutations in genes related to hereditary spastic paraplegia, two with mutations in genes related to Aicardi-Goutières syndrome, three with mutations in genes related to developmental/epileptic encephalopathies, and one with a mutation in the PGK1 gene. The accurate and precise patients' selection, the use of a high-throughput genetic platform, the selection of adequate target genes, and the application of rigorous criteria for the clinical interpretation are the most important elements for a good diagnostic performance. Based on our findings, next-generation sequencing should be considered in patients with cryptogenic CP as the first line of genetic workup. IMPACT: Sequencing techniques in CP of uncertain etiology provides a diagnostic yield of 55%. The appropriate selection of cases optimizes the diagnostic yield. NGS facilitate better understanding of new phenotypes of certain genetic diseases.

Implementation of massive sequencing in the genetic diagnosis of hereditary cancer syndromes: diagnostic performance in the Hereditary Cancer Programme of the Valencia Community (FamCan-NGS).

BACKGROUND: Approximately 5 to 10% of all cancers are caused by inherited germline mutations, many of which are associated with different Hereditary Cancer Syndromes (HCS). In the context of the Program of Hereditary Cancer of the Valencia Community, individuals belonging to specific HCS and their families receive genetic counselling and genetic testing according to internationally established guidelines. The current diagnostic approach is based on sequencing a few high-risk genes related to each HCS; however, this method is time-consuming, expensive and does not achieve a confirmatory genetic diagnosis in many cases. This study aims to test the level of improvement offered by a Next Generation Sequencing (NGS) gene-panel compared to the standard approach in a diagnostic reference laboratory setting. METHODS: A multi-gene NGS panel was used to test a total of 91 probands, previously classified as non-informative by analysing the high-risk genes defined in our guidelines. RESULTS: Nineteen deleterious mutations were detected in 16% of patients, some mutations were found in already-tested high-risk genes (BRCA1, BRCA2, MSH2) and others in non-prevalent genes (RAD51D, PALB2, ATM, TP53, MUTYH, BRIP1). CONCLUSIONS: Overall, our findings reclassify several index cases into different HCS, and change the mutational status of 14 cases from non-informative to gene mutation carriers. In conclusion, we highlight the necessity of incorporating validated multi-gene NGS panels into the HCSs diagnostic routine to increase the performance of genetic diagnosis.

TAF1 Variants Are Associated with Dysmorphic Features, Intellectual Disability, and Neurological Manifestations.

We describe an X-linked genetic syndrome associated with mutations in TAF1 and manifesting with global developmental delay, intellectual disability (ID), characteristic facial dysmorphology, generalized hypotonia, and variable neurologic features, all in male individuals. Simultaneous studies using diverse strategies led to the identification of nine families with overlapping clinical presentations and affected by de novo or maternally inherited single-nucleotide changes. Two additional families harboring large duplications involving TAF1 were also found to share phenotypic overlap with the probands harboring single-nucleotide changes, but they also demonstrated a severe neurodegeneration phenotype. Functional analysis with RNA-seq for one of the families suggested that the phenotype is associated with downregulation of a set of genes notably enriched with genes regulated by E-box proteins. In addition, knockdown and mutant studies of this gene in zebrafish have shown a quantifiable, albeit small, effect on a neuronal phenotype. Our results suggest that mutations in TAF1 play a critical role in the development of this X-linked ID syndrome.

High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing.

BACKGROUND: Intellectual disability is a very complex condition where more than 600 genes have been reported. Due to this extraordinary heterogeneity, a large proportion of patients remain without a specific diagnosis and genetic counselling. The need for new methodological strategies in order to detect a greater number of mutations in multiple genes is therefore crucial. METHODS: In this work, we screened a large panel of 1256 genes (646 pathogenic, 610 candidate) by next-generation sequencing to determine the molecular aetiology of syndromic intellectual disability. A total of 92 patients, negative for previous genetic analyses, were studied together with their parents. Clinically relevant variants were validated by conventional sequencing. RESULTS: A definitive diagnosis was achieved in 29 families by testing the 646 known pathogenic genes. Mutations were found in 25 different genes, where only the genes KMT2D, KMT2A and MED13L were found mutated in more than one patient. A preponderance of de novo mutations was noted even among the X linked conditions. Additionally, seven de novo probably pathogenic mutations were found in the candidate genes AGO1, JARID2, SIN3B, FBXO11, MAP3K7, HDAC2 and SMARCC2. Altogether, this means a diagnostic yield of 39% of the cases (95% CI 30% to 49%). CONCLUSIONS: The developed panel proved to be efficient and suitable for the genetic diagnosis of syndromic intellectual disability in a clinical setting. Next-generation sequencing has the potential for high-throughput identification of genetic variations, although the challenges of an adequate clinical interpretation of these variants and the knowledge on further unknown genes causing intellectual disability remain to be solved.

De novo mutations in genes of mediator complex causing syndromic intellectual disability: mediatorpathy or transcriptomopathy?

BACKGROUND: Mutations in the X-linked gene MED12 cause at least three different, but closely related, entities of syndromic intellectual disability. Recently, a new syndrome caused by MED13L deleterious variants has been described, which shows similar clinical manifestations including intellectual disability, hypotonia, and other congenital anomalies. METHODS: Genotyping of 1,256 genes related with neurodevelopment was performed by next-generation sequencing in three unrelated patients and their healthy parents. Clinically relevant findings were confirmed by conventional sequencing. RESULTS: Each patient showed one de novo variant not previously reported in the literature or databases. Two different missense variants were found in the MED12 or MED13L genes and one nonsense mutation was found in the MED13L gene. CONCLUSION: The phenotypic consequences of these mutations are closely related and/or have been previously reported in one or other gene. Additionally, MED12 and MED13L code for two closely related partners of the mediator kinase module. Consequently, we propose the concept of a common MED12/MED13L clinical spectrum, encompassing Opitz-Kaveggia syndrome, Lujan-Fryns syndrome, Ohdo syndrome, MED13L haploinsufficiency syndrome, and others.

Pure duplication of 19p13.3 in three members of a family with intellectual disability and literature review. Definition of a new microduplication syndrome.

This paper describes the presence of an interstitial pure duplication of 19p13.3 (4.95 Mb) in a patient with intellectual disability studied by array-CGH which was initially considered as a de novo alteration. The discovery of the same chromosomal alteration in a first-degree cousin of this patient led us to investigate the presence of insertional translocations, which were consequently found in three family generations. The same duplication was found in three intellectually disabled patients and among the translocation carrier family members a very high incidence of miscarriages are reported. A review of other published cases has allowed us to find three other patients with a similar pure duplication, all of them sharing some common clinical findings such as intrauterine growth retardation, microcephaly, motor and speech delay, moderate to severe intellectual disability, and dysmorphic features. These findings allow us to suggest the presence of a new microduplication syndrome in chromosomal region 19p13.3.

A novel missense mutation in the NSDHL gene identified in a Lithuanian family by targeted next-generation sequencing causes CK syndrome.

The NSDHL gene encodes 3ß-hydroxysteroid dehydrogenase involved in one of the later steps of the cholesterol biosynthetic pathway. Mutations in this gene can cause CHILD syndrome (OMIM 308050) and CK syndrome (OMIM 300831). CHILD syndrome is an X-linked dominant, male lethal disorder caused by mutations in the NSDHL gene that result in the loss of the function of the NSDHL protein. CK syndrome is an allelic X-linked recessive disorder. So far, 13 patients with CK syndrome from two families have been reported on. We present a new five-generation family with affected males manifesting clinical features of CK syndrome. Next generation sequencing was targeted to a custom panel of 542 genes with known or putative implication on intellectual disability. Missense mutation p.Gly152Asp was identified in the NSDHL gene in the DNA sample of the affected male. Mutation carrier status was confirmed for all the obligate carriers in the family. The clinical features of the affected males in the family manifested as weak fetal movements, severe intellectual disability, seizures, spasticity, atrophy of optic discs, microcephaly, plagiocephaly, skeletal abnormalities, and minor facial anomalies, including a high nasal bridge, strabismus, and micrognathia. A highly significant preferential transmission of the mutation was observed in this and previous families segregating CK syndrome. Our report expands the clinical spectrum of this syndrome to include weak fetal movements, spasticity, and plagiocephaly, and transmission ratio distortion. The various findings in these patients increase our understanding of the diversity of the clinical presentation of cholesterol biosynthesis disorders.

Novel mutations of NFIX gene causing Marshall-Smith syndrome or Sotos-like syndrome: one gene, two phenotypes.

BACKGROUND: Only 15 point mutations in NFIX gene have been reported so far, nine of them cause the Marshall-Smith syndrome (MSS) and the remaining mutations lead to an overgrowth disorder with a less severe phenotype, defined as Sotos-like. METHODS: The clinical findings in three patients with MSS and two patients with a Sotos-like phenotype are presented. Analysis of the NFIX gene was performed both by conventional or next-generation sequencing. RESULTS: Five de novo mutations in NFIX gene were identified, four of them not previously reported. Two frameshift mutations and a donor-splice one caused MSS, while two missense mutations in the DNA binding/dimerisation domain entailed an overgrowth syndrome with some clinical features resembling Sotos syndrome, accompanied by a marfanoid habitus, very low BMI, long narrow face, or arachnodactyly. CONCLUSION: Marshall-Smith mutations are scattered through exons 6-10 of NFIX gene, while most point mutations causing an overgrowth syndrome are clustered in exon 2. Clinical features of this overgrowth syndrome may well be considered an intermediate phenotype between Sotos and Marfan syndromes.

Duplication at Xq13.3-q21.1 with syndromic intellectual disability, a probable role for the ATRX gene.

Here we report on two unrelated male patients with syndromic intellectual disability (ID) due to duplication at Xq13.3-q21.1, a region of about 6 Mb and 25 genes. Among these, the most outstanding is ATRX, the causative gene of X-linked alpha-thalassemia/mental retardation. ATRX belongs to the growing list of genes implied in chromatin remodeling causing ID. Many these genes, such as MECP2, are dose-sensitive so that not only deletions and point mutations, but also duplications cause ID. Both patients have severe ID, absent expressive speech, early hypotonia, behavior problems (hyperactivity, repetitive self-stimulatory behavior), postnatal growth deficiency, microcephaly, micrognathia, cryptorchidism, low-set, posteriorly angulated ears, and downslanting palpebral fissures. These findings are also usually present among patients with loss-of-function mutations of the ATRX gene. Completely skewed X inactivation was observed in the only informative carrier mother, a constant finding among female carriers of inactivating point mutations of this gene. Participation of other duplicated genes cannot be excluded; nevertheless we propose that the increased dosage of ATRX is the major pathogenic mechanism of this X-linked disorder, a syndrome reminiscent of MECP2 duplication.

Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 2 Caused by a Novel PIGA Variant Not Associated with a Skewed X-Inactivation Pattern.

Germline variants in the phosphatidylinositol glycan class A (PIGA) gene, which is involved in glycosylphosphatidylinositol (GPI) biosynthesis, cause multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) with X-linked recessive inheritance. The available literature has described a pattern of almost 100% X-chromosome inactivation in mothers carrying PIGA variants. Here, we report a male infant with MCAHS2 caused by a novel PIGA variant inherited from his mother, who has a non-skewed pattern of X inactivation. Phenotypic evidence supporting the pathogenicity of the variant was obtained by flow-cytometry tests. We propose that the assessment in neutrophils of the expression of GPI-anchored proteins (GPI-APs), especially CD16, should be considered in cases with variants of unknown significance with random X-inactivation in carrier mothers in order to clarify the pathogenic role of PIGA or other gene variants linked to the synthesis of GPI-APs.

Analysis of SNP rs16754 of WT1 gene in a series of de novo acute myeloid leukemia patients.

The single nucleotide polymorphism (SNP) rs16754 of the WT1 gene has been previously described as a possible prognostic marker in normal karyotype acute myeloid leukemia (AML) patients. Nevertheless, the findings in this field are not always reproducible in different series. One hundred and seventy-five adult de novo AML patients were screened with two different methods for the detection of SNP rs16754: high-resolution melting (HRM) and FRET hybridization probes. Direct sequencing was used to validate both techniques. The SNP was detected in 52 out of 175 patients (30 %), both by HRM and hybridization probes. Direct sequencing confirmed that every positive sample in the screening methods had a variation in the DNA sequence. Patients with the wild-type genotype (WT1(AA)) for the SNP rs16754 were significantly younger than those with the heterozygous WT1(AG) genotype. No other difference was observed for baseline characteristic or outcome between patients with or without the SNP. Both techniques are equally reliable and reproducible as screening methods for the detection of the SNP rs16754, allowing for the selection of those samples that will need to be sequenced. We were unable to confirm the suggested favorable outcome of SNP rs16754 in de novo AML.

Germline variant in Ctcf links mental retardation to Wilms tumor predisposition.

CTCF germline mutations have been related to MRD21. We report the first bilateral Wilms tumor suffered by a MRD21 patient carrying an unreported CTCF missense variant in a zinc finger domain of CTCF protein. We found that germline heterozygous variant I446K became homozygous in the tumor due to a loss of heterozygosity rearrangement affecting the whole q arm on chromosome 16. Our findings propose CTCF I446K variant as a link between MRD21 and Wilms tumor predisposition.

Hypermethylation of apoptotic genes as independent prognostic factor in neuroblastoma disease.

Neuroblastoma (NB) is an embryonal tumour of neuroectodermal cells, and its prognosis is based on patient age at diagnosis, tumour stage and MYCN amplification, but it can also be classified according to their degree of methylation. Considering that epigenetic aberrations could influence patient survival, we studied the methylation status of a series of 17 genes functionally involved in different cellular pathways in patients with NB and their impact on survival. We studied 82 primary NB tumours and we used methylation-specific-PCR to perform the epigenetic analysis. We evaluated the putative association among the evidence of hypermethylation with the most important NB prognostic factors, as well as to determine the relationship among methylation, clinical classification and survival. CASP8 hypermethylation showed association with relapse susceptibility and, TMS1 and APAF1 hypermethylation are associated with bad prognosis and showed high influence on NB overall survival. Hypermethylation of apoptotic genes has been identified as a good candidate of prognostic factor. We propose the simultaneous analysis of hypermethylation of APAF1, TMS1 and CASP8 apoptotic genes on primary NB tumour as a good prognostic factor of disease progression.

Intronic mutations affecting splicing of MBTPS2 cause ichthyosis follicularis, alopecia and photophobia (IFAP) syndrome.

Ichthyosis follicularis, alopecia and photophobia (IFAP) syndrome is an X-linked genodermatosis with congenital atrichia being the most prominent feature. Recently, we have shown that functional deficiency of MBTPS2 (membrane-bound transcription factor protease site 2) - a zinc metalloprotease essential for cholesterol homeostasis and endoplasmic reticulum stress response - causes the disease. Here, we present results obtained by analysing two intronic MBTPS2 mutations, c.671-9T>G and c.225-6T>A, using in silico and cell-based splicing assays. Accordingly, the c.225-6T>A transversion generated a new splice acceptor site, which caused extension of exon 3 by four bases and subsequently introduced a premature stop codon. Both, minigene experiments and RT-PCR analysis with patient-derived mRNA, demonstrated that the c.671-9T>G mutation resulted in skipping of exon 6, most likely because of disruption of the polypyrimidin tract or a putative intronic splicing enhancer (ISE). Our combined biocomputational and experimental analysis strongly suggested that both intronic alterations are disease-causing mutations.

Prevalence of pathogenic copy number variants among children conceived by donor oocyte.

Development of assisted reproductive technologies to address infertility has favored the birth of many children in the last years. The majority of children born with these treatments are healthy, but some concerns remain on the safety of these medical procedures. We have retrospectively analyzed both the fertilization method and the microarray results in all those children born between 2010 and 2019 with multiple congenital anomalies, developmental delay and/or autistic spectrum disorder (n = 486) referred for array study in our center. This analysis showed a significant excess of pathogenic copy number variants among those patients conceived after in vitro fertilization with donor oocyte with respect to those patients conceived by natural fertilization (p = 0.0001). On the other hand, no significant excess of pathogenic copy number variants was observed among patients born by autologous oocyte in vitro fertilization. Further studies are necessary to confirm these results and in order to identify the factors that may contribute to an increased risk of genomic rearrangements, as well as consider the screening for genomic alterations after oocyte donation in prenatal diagnosis.

Retinoblastoma and mosaic 13q deletion: a case report.

BACKGROUND: Patients with 13q-syndrome are at risk of retinoblastoma when the RB1 gene, located in the chromosomal band 13q14.2, is deleted. This syndrome is frequently associated with congenital malformations and developmental delay, although these signs could be mild. Mosaic 13q-deletion patients have been previously reported in the literature; their phenotype is variable, and they may not be recognized. CASE PRESENTATION: Retinoblastoma diagnosed in a child with 13q-mosaicism confirmed in blood, oral mucosa, healthy retina and retinoblastoma. A second RB1 hit is present exclusively in the retinoblastoma sample (RB1 c.958C>T p.Arg320Ter). Other detected molecular events in retinoblastoma are 6p12.3pter gain and 6q25.3qter loss. Clinical examination is unremarkable except for clinodactyly of the right fifth finger. DISCUSSION AND CONCLUSIONS: We describe a case of mosaic 13q deletion syndrome affected by retinoblastoma. Molecular data obtained from the tumor analysis are similar to previous data available about this malignancy. High clinical suspicion is essential for an adequate diagnosis of mosaic cases.

Germline Predisposition to Pediatric Cancer, from Next Generation Sequencing to Medical Care.

Knowledge about genetic predisposition to pediatric cancer is constantly expanding. The categorization and clinical management of the best-known syndromes has been refined over the years. Meanwhile, new genes for pediatric cancer susceptibility are discovered every year. Our current work shares the results of genetically studying the germline of 170 pediatric patients diagnosed with cancer. Patients were prospectively recruited and studied using a custom panel, OncoNano V2. The well-categorized predisposing syndromes incidence was 9.4%. Likely pathogenic variants for predisposition to the patient's tumor were identified in an additional 5.9% of cases. Additionally, a high number of pathogenic variants associated with recessive diseases was detected, which required family genetic counseling as well. The clinical utility of the Jongmans MC tool was evaluated, showing a high sensitivity for detecting the best-known predisposing syndromes. Our study confirms that the Jongmans MC tool is appropriate for a rapid assessment of patients; however, the updated version of Ripperger T criteria would be more accurate. Meaningfully, based on our findings, up to 9.4% of patients would present genetic alterations predisposing to cancer. Notably, up to 20% of all patients carry germline pathogenic or likely pathogenic variants in genes related to cancer and, thereby, they also require expert genetic counseling. The most important consideration is that the detection rate of genetic causality outside Jongmans MC et al. criteria was very low.

Recurrent Metabolic Alkalosis in a Cystic Fibrosis Patient: Coexistence with Congenital Chloride Diarrhea.

Metabolic alkalosis is uncommon in infancy. Cystic fibrosis (CF) patients can develop dehydration because of sweat salt or gastrointestinal losses; with the correct salt supplementation, the electrolyte alterations can be reversed. Here, we present a CF patient with recurrent metabolic alkalosis, initially oriented as pseudo-Bartter's syndrome. However, despite accurate treatment, patient needed daily intravenous fluids to maintain homeostasis. An extended study was made, including a urine study that could rule out Bartter's diagnosis. Finally, after a complementary test that included electrolyte stools study and genetic analysis, congenital chloride diarrhea could be diagnosed.