Search

Neurodevelopmental syndromes due to copy number variation are well-known clinical entities. While the numerical variation of gene-harboring regions has been widely investigated at both molecular and clinical levels, much less is understood about unbalanced expression of long noncoding RNAs. Few studies have been performed on the clinical consequences of such unbalanced expression. Heterozygous deletions of NRXN1 have been well described to cause neuropsychological features. Heterozygous deletion of adjacent long noncoding RNA AK127244, either isolated or combined with partial NRXN1 deletion, was recently reported in association with neurodevelopmental delay. In our retrospective study, we analyze a bicentric cohort of 4 individuals, comprising 2 siblings, which bear an isolated heterozygous deletion in long noncoding RNA AK127244 and present with nonsyndromic neurodevelopmental delay.

Type 1 Chiari malformation (C1M) is characterized by cerebellar tonsillar herniation of 3–5 mm or more, the frequency of which is presumably much higher than one in 1000 births, as previously believed. Its etiology remains undefined, although a genetic basis is strongly supported by C1M presence in numerous genetic syndromes associated with different genes. Whole-exome sequencing (WES) in 51 between isolated and syndromic pediatric cases and their relatives was performed after confirmation of the defect by brain magnetic resonance image (MRI). Moreover, in all the cases showing an inherited candidate variant, brain MRI was performed in both parents and not only in the carrier one to investigate whether the defect segregated with the variant. More than half of the variants were Missense and belonged to the same chromatin-remodeling genes whose protein truncation variants are associated with severe neurodevelopmental syndromes. In the remaining cases, variants have been detected in genes with a role in cranial bone sutures, microcephaly, neural tube defects, and RASopathy. This study shows that the frequency of C1M is widely underestimated, in fact many of the variants, in particular those in the chromatin-remodeling genes, were inherited from a parent with C1M, either asymptomatic or with mild symptoms. In addition, C1M is a Mendelian trait, in most cases inherited as dominant. Finally, we demonstrate that modifications of the genes that regulate chromatin architecture can cause localized anatomical alterations, with symptoms of varying degrees. Electronic supplementary material The online version of this article (10.1007/s00439-020-02231-6) contains supplementary material, which is available to authorized users.

Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia.

Marfan syndrome (MFS) and Loeys–Dietz syndrome type 4 (LDS4) are two hereditary connective tissue disorders. MFS displays ectopia lentis as a distinguishing, characterising feature, and thoracic aortic ectasia, aneurysm, dissection, and systemic features as manifestations overlapping with LDS4. LDS4 is characterised by the presence of hypertelorism, cleft palate and/or bifid uvula, with possible ectasia or aneurysms in other arteries. The variable age of onset of clinical manifestations makes clinical diagnosis more difficult. In this study, we report the case of a patient with Marfan syndrome diagnosed at our centre at the age of 33 on the basis of typical clinical manifestations of this syndrome. At the age of 38, the appearance of ectasia of the left common iliac artery and tortuosity of the iliac arteries suggested the presence of LDS4. Next Generation Sequencing (NGS) analysis, followed by Array-CGH, allowed the detection of a novel chromosomal deletion including the entire TGFB2 gene, confirming not only the clinical suspicion of LDS4, but also the clinical phenotype associated with the haploinsufficiency mechanism, which is, in turn, associated with the deletion of the entire gene. The same mutation was detected in the two young sons. This emblematic case confirms that we must be very careful in the differential diagnosis of these two pathologies, especially before the age of 40, and that, in young subjects suspected to be affected by MFS in particular, we must verify the diagnosis, extending genetic analysis, when necessary, to the search for chromosomal alterations. Recently, ectopia lentis has been reported in a patient with LDS4, confirming the tight overlap between the two syndromes. An accurate revision of the clinical parameters both characterising and overlapping the two pathologies is highly desirable.

Cancer predisposition syndromes (CPS) result from germline pathogenic variants, and they are increasingly recognized in the etiology of many pediatric cancers. Herein, we report the genetic/genomic analysis of 40 pediatric patients enrolled from 2016 to 2018. Our diagnostic workflow was successful in 50% of screened cases. Overall, the proportion of CPS in our case series is 10.9% (20/184) of enrolled patients. Interestingly, 12.5% of patients achieved a conclusive diagnosis through the analysis of chromosomal imbalance. Indeed, we observed germline microdeletions/duplications of regions encompassing cancer-related genes in 50% of patients undergoing array-CGH: EIF3H duplication in a patient with infantile desmoplastic astrocytoma and low-grade Glioma; SLFN11 deletion, SOX4 duplication, and PARK2 partial deletion in three neuroblastoma patients; a PTPRD partial deletion in a child diagnosed with glioblastoma multiforme. Finally, we identified two cases due to DICER1 germline mutations.

Background Mutations in lysyl-tRNA synthetase (KARS1), an enzyme that charges tRNA with the amino acid lysine in both the cytoplasm and mitochondria, have been associated thus far with autosomal recessive Charcot–Marie–Tooth type CMTRIB, hearing loss type DFNB89, and mitochondrial encephalohepatopathy (MEH) featuring neurodevelopmental disorders with microcephaly, white matter changes, and cardiac and hepatic failure in less than 30 patients. Case presentation We report the clinical, biochemical and molecular findings of a 14-month-old girl with severe MEH compatible clinical features, profound sensorineural hearing loss, leopard spot retinopathy, pancytopenia, and advanced liver disease with portal hypertension leading to death at the age of 30 months. Conclusions Whole exome sequencing identified two rare variants in KARS1 gene. Our report expands the allelic and clinical features of tRNA synthase disorders. Moreover, with our report we confirm the usefulness of WES as first tier diagnostic method in infants with complex multisystem phenotypes.

Background The diversity in the clinical course of COVID-19 has been related to differences in innate and adaptative immune response mechanisms. Natural killer (NK) lymphocytes are critical protagonists of human host defense against viral infections. It would seem that reduced circulating levels of these cells have an impact on COVID-19 progression and severity. Their activity is strongly regulated by killer-cell immuno-globulin-like receptors (KIRs) expressed on the NK cell surface. The present study’s focus was to investigate the impact of KIRs and their HLA Class I ligands on SARS-CoV-2 infection. Methods KIR gene frequencies, KIR haplotypes, KIR ligands and combinations of KIRs and their HLA Class I ligands were investigated in 396 Sardinian patients with SARS-CoV-2 infection. Comparisons were made between 2 groups of patients divided according to disease severity: 240 patients were symptomatic or paucisymptomatic (Group A), 156 hospitalized patients had severe disease (Group S). The immunogenetic characteristics of patients were also compared to a population group of 400 individuals from the same geographical areas. Results Substantial differences were obtained for KIR genes, KIR haplotypes and KIR-HLA ligand combinations when comparing patients of Group S to those of Group A. Patients in Group S had a statistically significant higher frequency of the KIR A/A haplotype compared to patients in Group A [34.6% vs 23.8%, OR = 1.7 (95% CI 1.1–2.6); P = 0.02, Pc = 0.04]. Moreover, the KIR2DS2/HLA C1 combination was poorly represented in the group of patients with severe symptoms compared to those of the asymptomatic-paucisymptomatic group [33.3% vs 50.0%, OR = 0.5 (95% CI 0.3–0.8), P = 0.001, Pc = 0.002]. Multivariate analysis confirmed that, regardless of the sex and age of the patients, the latter genetic variable correlated with a less severe disease course [ORM = 0.4 (95% CI 0.3–0.7), PM = 0.0005, PMC = 0.005]. Conclusions The KIR2DS2/HLA C1 functional unit resulted to have a strong protective effect against the adverse outcomes of COVID-19. Combined to other well known factors such as advanced age, male sex and concomitant autoimmune diseases, this marker could prove to be highly informative of the disease course and thus enable the timely intervention needed to reduce the mortality associated with the severe forms of SARS-CoV-2 infection. However, larger studies in other populations as well as experimental functional studies will be needed to confirm our findings and further pursue the effect of KIR receptors on NK cell immune-mediated response to SARS-Cov-2 infection.

The heterogeneous group of mitochondrial disorders (MDs) is by far the largest class of inborn errors of metabolism, having a collective incidence of 1.6 in 5000.1 Over the past decade, routine clinical use of next-generation sequencing (NGS) has allowed a considerable number of cases to receive a precise molecular diagnosis. At present, however, NGS approaches, including targeted multigene panels and exome sequencing, still fail to achieve molecular definition in about half of patients with MD with a confirmed biochemical and clinical diagnosis.2 These unresolved cases could be due to new (‘hyper-rare’) genes/loci; alternatively, they could be linked to limits of current NGS strategies: indeed, mosaicisms requiring high sequencing depth for detection, genomic rearrangements including small and large deletions/insertions ( 50 bp, respectively), and structural changes such as chromosomal rearrangements (inversions, translocations, deletions, duplications) all usually escape traditional NGS analysis pipelines. The chronic limited use of whole-genome sequencing in diagnostic settings may contribute to the substantial number of unresolved cases. The presence of repeat expansions and variants in deep intronic and regulatory regions2 may also provide an explanation for ‘missing heritability’. Although relatively rare, UPD might explain the unsolved transmission of homozygous mutations or aberrant patterns of imprinting,3 even in MDs. Here, we describe the cases of two children, each with a clinical diagnosis of MD, in whom chromosomal microarray analysis (CMA) allowed detection of UPD and helped to substantiate a homozygous variant identified by NGS. This study was conducted in accordance with Italian National Health System guidelines and the Declaration of Helsinki. Patient 1 is a child who, when first seen by us, presented with generalised tonic-clonic seizures, hypotonia and …

Background Stickler Syndrome is a rare connective tissue disorder, characterized by clinical, and genetic heterogeneity. The clinical expression is highly variable, including moderate to severe myopia in childhood, hearing loss, facial dysmorphic features, cleft palate, and early osteoarthritis. COL2A1, COL11A1, and COL11A2 mutations account of the majority of autosomal dominant Stickler Syndrome and, in particular, a heterozygous mutation in COL11A1 gene is identified in about 10 to 20% of Stickler Syndrome patients. Methods Herein, we report a case of an 8‐year‐ old child with Stickler Syndrome, presenting with early‐onset of myopia with vitreal abnormalities, facial dysmorphic characteristics, and mild hearing loss later in childhood. To identify the underlying genetic cause, Whole Exome Sequencing was carried out for COL11A1 gene. Results A novel de novo heterozygous splice site variant (NM_001854: c.1845 + 5G> C) of the COL11A1 gene, which had not been previously reported, was identified by Whole Exome Sequencing. Conclusion We reported a novel COL11A1 mutation in a child with Stickler Syndrome presenting a phenotype of early‐onset of ocular anomalies and mild hearing loss later in childhood. Our findings confirm the variability of the expression of the disease, even in the contest of the same gene‐related disorder, thus, contributing to improve the knowledge on clinical and molecular basis of this rare disease., A novel COL11A1 mutation identified by Whole Genome Sequencing, confirming the diagnosis of Stickler Syndrome in a child. Clinical features included early‐onset of myopia with vitreal abnormalities, facial dysmorphic characteristics and mild hearing loss later in childhood.

Background COVID‐19 outbreak prompted health centres to reorganize their clinical and surgical activity. In this paper, we show how medical genetics department's activity, in our tertiary pediatric hospital, has changed due to pandemic. Methods We stratified all our scheduled visits, from March 9th through April 30th, and assessed case‐by‐case which genetic consultations should be maintained as face‐to‐face visit, or postponed/switched to telemedicine. Results Out of 288 scheduled appointments, 60 were prenatal consultations and 228 were postnatal visits. We performed most of prenatal consultations as face‐to‐face visits, as women would have been present in the hospital to perform other procedures in addition to our consult. As for postnatal care, we suspended all outpatient first visits and opted for telemedicine for selected follow‐up consultations: interestingly, 75% of our patients’ parents revealed that they would have cancelled the appointment themselves for the fear to contract an infection. Conclusions Spread of COVID‐19 in Italy forced us to change our working habits. Given the necessity to optimize healthcare resources and minimize the risk of in‐hospital infections, we experienced the benefits of telegenetics. Current pandemic made us familiar with telemedicine, laying the foundations for its application to deal with the increasing number of requests in clinical genetics., In this paper, we show how medical genetics department's activity, in our tertiary pediatric hospital, has changed due to COVID‐19 pandemic. Given the necessity to optimize healthcare resources and minimize the risk of in‐hospital infections, we experienced the benefits of telegenetics. Current pandemic made us familiar with telemedicine, laying the foundations for its application to deal with the increasing number of requests in clinical genetics.

Background and Aims Monogenic inherited diseases are an underestimated but important cause of chronic kidney disease (CKD). Despite the increasing knowledge about their inheritance, they are identified in fewer than 10% of CKD patients. Among monogenic causes of CKD, polycystic kidney diseases (PKD) represent a group of disorders with a clinical and genetic heterogeneity and a variable phenotype (from early manifestations during pregnancy or childhood to the absence of clinical manifestations until adulthood). We here present a PKD family with 5 members displaying the disease (Figure 1). Method We revised patients clinical history, laboratory data and imaging exams. An accurate family history was recovered. Blood samples were collected from living patients to perform an exome analysis using next generation sequencing to analyze all possible genes involved in PKD. Results Each patient presented a clinical history of nephrolithiasis, a variable degree of CKD (from normal kidney function to renal transplant), urinary and blood parameters compatible with distal renal tubular acidosis (hypercalciuria, low urinary citrate excretion and hypokalemia). Imaging exams showed several renal cysts in the medulla or at the cortico-medullary junction with intracystic calcifications (Figure 2). Some patients displayed also large cortical renal cysts and also hepatic and pancreatic cysts. The genetic analysis performed using next generation sequencing in four patients showed a new mutation on the gene DNAJB11; the mutation introduced a stop codon and was identified as pathogenic by prediction programs. It was present in heterozygosity in all subjects and had an autosomal dominant pattern of inheritance. Conclusion DNAJB11 mutations were reported as a cause of PKD for the first time in 2018 by Cornec-Le-Gall and colleagues. The nephropathy derived from these mutations need to be differentiated from ADPKD (autosomal dominant polycystic kidney disease), from ADTKD (autosomal dominant tubulointerstitial kidney disease) and from medullary sponge kidney. DNAJB11 encodes a co-chaperone of the endoplasmic reticulum (ER) also called ERdj3. It is part of the HSP40 protein family and plays a central role in both intracellular and extracellular proteomic homeostasis (proteostasis). In the intracellular compartment it acts as a co-chaperone in the pathway of the unfolded protein response (UPR) in which it binds misfolded proteins which have to be secreted and activates BiP an HSP70 of ER whose function is to correct the misfolding. In the kidney it is mainly expressed in the thick ascending limb of Henle’s loop, in the distal tubule and in the collecting duct justifying the clinical presentation of nephropathy and differentiating the disease from ADPKD (Figure 3).

We investigated 52 cases of de novo unbalanced translocations, consisting in a terminally deleted or inverted-duplicated deleted (inv-dup del) 46th chromosome to which the distal portion of another chromosome or its opposite end was transposed. Array CGH, whole-genome sequencing, qPCR, FISH, and trio genotyping were applied. A biparental origin of the deletion and duplication was detected in 6 cases, whereas in 46, both imbalances have the same parental origin. Moreover, the duplicated region was of maternal origin in more than half of the cases, with 25% of them showing two maternal and one paternal haplotype. In all these cases, maternal age was increased. These findings indicate that the primary driver for the occurrence of the de novo unbalanced translocations is a maternal meiotic non-disjunction, followed by partial trisomy rescue of the supernumerary chromosome present in the trisomic zygote. In contrast, asymmetric breakage of a dicentric chromosome, originated either at the meiosis or postzygotically, in which the two resulting chromosomes, one being deleted and the other one inv-dup del, are repaired by telomere capture, appears at the basis of all inv-dup del translocations. Notably, this mechanism also fits with the origin of some simple translocations in which the duplicated region was of paternal origin. In all cases, the signature at the translocation junctions was that of non-homologous end joining (NHEJ) rather than non-allelic homologous recombination (NAHR). Our data imply that there is no risk of recurrence in the following pregnancies for any of the de novo unbalanced translocations we discuss here.

Whole exome sequencing (WES) has made the identification of causative SNVs/InDels associated with rare Mendelian conditions increasingly accessible. Incorporation of softwares allowing CNVs detection into the WES bioinformatics pipelines may increase the diagnostic yield. However, no standard protocols for this analysis are so far available and CNVs in non-coding regions are totally missed by WES, in spite of their possible role in the regulation of the flanking genes expression. So, in a number of cases the diagnostic workflow contemplates an initial investigation by genomic arrays followed, in the negative cases, by WES. The opposite workflow may also be applied, according to the familial segregation of the disease. We show preliminary results for a diagnostic application of a single next generation sequencing panel permitting the concurrent detection of LOH and variations in sequences and copy number. This approach allowed us to highlight compound heterozygosity for a CNV and a sequence variant in a number of cases, the duplication of a non-coding region responsible for sex reversal, and a whole-chromosome isodisomy causing reduction to homozygosity for a WFS1 variant. Moreover, the panel enabled us to detect deletions, duplications, and amplifications with sensitivity comparable to that of the most widely used array-CGH platforms.

Prenatal diagnosis of skeletal dysplasias is particularly difficult for many reasons and differentiating these disorders in the prenatal period can be challenging because they are rare and many of the ultrasound findings are not necessarily pathognomonic for a specific disorder. The diagnosis is often made just after birth or exitus. The prenatal diagnosis of osteochondrodysplasias is based predominantly upon fetal ultrasound findings and it focuses substantially on the possible lethality of the disorder, without always being able to find a specific name for the disorder. Metatropic dysplasia is a rare osteochondrodysplasia due to mutations in the TRPV4 gene: TRPV4 is a cation channel, non-selectively permeable to calcium, encoded by a gene on chromosome 12q24.11; it is widely expressed and involved in many different physiological processes through responses to several different stimuli (physical, chemical, and hormonal) in ciliated epithelial cells. The exact incidence of this disorder is not known, however less than a hundred cases have been reported at present, with only two prenatal reports but without any reference to the molecular test. We describe the first report of molecular diagnosis of metatropic dysplasia carried out in prenatal diagnosis: the molecular testing of the TRPV4 (transient receptor potential cation channel, subfamily V, member 4, MIM *605427) gene in our case, in fact, detected a causative variant, confirming the diagnostic suspicion, which was made possible thanks also to the utilization of MRI and CT scan. In our case different imaging methods together with the close cooperation of a multidisciplinary team and test availability, allowed an accurate diagnosis.

PTEN hamartoma tumor syndrome (PHTS) refers to a group of clinical conditions caused by germline mutations in the PTEN tumor suppressor gene. Increasing evidence has documented that PHTS may be associated with a broader spectrum of structural brain abnormalities, including dysplastic gangliocytoma of the cerebellum, brain tumors, vascular malformations, white matter abnormalities, dilated perivascular spaces and cortical dysplasia. We report a PTEN-mutated child showing macrocephaly, mild intellectual disability and epilepsy symptomatic of right occipital polymicrogyria, who also developed Chiari I Malformation (CIM) that repeatedly required surgical correction. We suppose that the association between PHTS and CIM could be not coincidental, thus extending the spectrum of neurological manifestations of PHTS and highlighting the role of brain MRI in the management of PHTS patients. We suggest that genes within the RAS-MAPK and PI3-AKT pathways might have a significant role in the pathogenesis of CIM in such patients.

BackgroundThe regulation of the chromatin state by epigenetic mechanisms plays a central role in gene expression, cell function, and maintenance of cell identity. Hereditary disorders of chromatin regulation are a group of conditions caused by abnormalities of the various components of the epigenetic machinery, namely writers, erasers, readers, and chromatin remodelers. Although neurological dysfunction is almost ubiquitous in these disorders, the constellation of additional features characterizing many of these genes and the emerging clinical overlap among them indicate the existence of a community of syndromes. The introduction of high-throughput next generation sequencing (NGS) methods for testing multiple genes simultaneously is a logical step for the implementation of diagnostics of these disorders.MethodsWe screened a heterogeneous cohort of 263 index patients by an NGS-targeted panel, containing 68 genes associated with more than 40 OMIM entries affecting chromatin function.ResultsThis strategy allowed us to identify clinically relevant variants in 87 patients (32%), including 30 for which an alternative clinical diagnosis was proposed after sequencing analysis and clinical re-evaluation.ConclusionOur findings indicate that this approach is effective not only in disorders with locus heterogeneity, but also in order to anticipate unexpected misdiagnoses due to clinical overlap among cognate disorders. Finally, this work highlights the utility of a prompt diagnosis in such a clinically and genetically heterogeneous group of disorders that we propose to group under the umbrella term of chromatinopathies.

Background: Biallelic pathogenic variants in MFRP and PRSS56 genes can be responsible for nanophthalmos (NO) or posterior microphthalmos (PM). This study describes detailed clinical and molecular f...

Correct diagnosis of Maturity-Onset Diabetes of the Young (MODY) is based on genetic tests requiring an appropriate subject selection by clinicians. Mutations in the insulin (INS) gene rarely occur in patients with MODY. This study is aimed at determining the genetic background and clinical phenotype in patients with suspected MODY. 34 patients with suspected MODY, negative for mutations in the GCK, HNF1α, HNF4α, HNF1β and PDX1 genes, were screened by next generation sequencing (NGS). A heterozygous INS mutation was identified in 4 members of the same family. First genetic tests performed identified two heterozygous silent nucleotide substitutions in MODY3/HNF1α gene. An ineffective attempt to suspend insulin therapy, administering repaglinide and sulphonylureas, was made. DNA was re-sequenced by NGS investigating a set of 102 genes. Genes implicated in the pathway of pancreatic β-cells, candidate genes for type 2 diabetes mellitus and genes causative of diabetes in mice were selected. A novel heterozygous variant in human preproinsulin INS gene (c.125T > C) was found in the affected family members. The new INS mutation broadens the spectrum of possible INS phenotypes. Screening for INS mutations is warranted not only in neonatal diabetes but also in MODYx patients and in selected patients with type 1 diabetes mellitus negative for autoantibodies. Subjects with complex diseases without a specific phenotype should be studied by NGS because Sanger sequencing is ineffective and time consuming in detecting rare variants.

Background Genetic polymorphisms in genes involved in pain modulation have been reported to be associated to opioid efficacy and safety in different clinical settings. Methods The association between COMT Val158Met polymorphism (rs4680) and the inter-individual differences in the response to opioid analgesic therapy was investigated in a cohort of 87 Italian paediatric patients receiving opioids for cancer pain (STOP Pain study). Furthermore, a systematic review of the association between opioid response in cancer patients and the COMT polymorphism was performed in accordance with the Cochrane Handbook and the Prisma Statement. Results In the 87 paediatric patients, pain intensity (total time needed to reach the lowest possible level) was significantly higher for G/G than A/G and A/A carriers (p-value = 0.042). In the 60 patients treated only with morphine, the mean of total dose to reach the same pain intensity was significantly higher for G/G than A/G and A/A carriers (p-value = 0.010). Systematic review identified five studies on adults, reporting that opioid dose (mg after 24 h of treatment from the first pain measurement) was higher for G/G compared to A/G and A/A carriers. Conclusions Present research suggests that the A allele in COMT polymorphism could be a marker of opioid sensitivity in paediatric cancer patients (STOP Pain), as well as in adults (Systematic Review), indicating that the polymorphism impact could be not age-dependent in the cancer pain context. Trial registration Registration number: CRD42017057831. Electronic supplementary material The online version of this article (10.1186/s12885-019-5310-4) contains supplementary material, which is available to authorized users.

Periventricular nodular heterotopia (PNH) is a brain malformation in which nodules of neurons are ectopically retained along the lateral ventricles. Genetic causes include FLNA abnormalities (classical X-linked PNH), rare variants in ARFGEF2, DCHS1, ERMARD, FAT4, INTS8, MAP1B, MCPH1, and NEDD4L, as well as several chromosomal abnormalities. We performed array-CGH in 106 patients with different malformations of cortical development (MCD) and looked for common pathways possibly involved in PNH. Forty-two patients, including two parent/proband couples, exhibited PNH associated or not with other brain abnormalities, 44 had polymicrogyria and 20 had rarer MCDs. We found an enrichment of either large rearrangements or cryptic copy number variants (CNVs) in PNH (15/42, 35.7%) vs polymicrogyria (4/44, 9.1%) (i.e., 5.6 times increased risk for PNH of carrying a pathogenic CNV). CNVs in seven genomic regions (2p11.2q12.1, 4p15, 14q11.2q12, 16p13.3, 19q13.33, 20q13.33, 22q11) represented novel, potentially causative, associations with PNH. Through in silico analysis of genes included in imbalances whose breakpoints were clearly detailed, we detected in 9/12 unrelated patients in our series and in 15/24 previously published patients, a significant (P

We studied by a whole genomic approach and trios genotyping, 12 de novo, nonrecurrent small supernumerary marker chromosomes (sSMC), detected as mosaics during pre- or postnatal diagnosis and associated with increased maternal age. Four sSMCs contained pericentromeric portions only, whereas eight had additional non-contiguous portions of the same chromosome, assembled together in a disordered fashion by repair-based mechanisms in a chromothriptic event. Maternal hetero/isodisomy was detected with a paternal origin of the sSMC in some cases, whereas in others two maternal alleles in the sSMC region and biparental haplotypes of the homologs were detected. In other cases, the homologs were biparental while the sSMC had the same haplotype of the maternally inherited chromosome. These findings strongly suggest that most sSMCs are the result of a multiple-step mechanism, initiated by maternal meiotic nondisjunction followed by postzygotic anaphase lagging of the supernumerary chromosome and its subsequent chromothripsis.

Hypohidrotic ectodermal dysplasia (HED) is a rare disease characterized by deficiency in development of structure derived from the ectoderm and is caused by mutations in the genes EDA, EDAR, or EDARADD. Phenotypes caused by mutations in these three may exhibit similar clinical features, explained by a common signaling pathway. Mutations in EDA gene cause X linked HED, which is the most common form. Mutations in EDAR and EDARADD genes cause autosomal dominant and recessive form of HED. The most striking clinical findings in HED are hypodontia, hypotrichosis and hypohidrosis that can lead to episodes of hyperthermia. We report on clinical findings in a child with HED with autosomal dominant inheritance pattern with a heterozygous mutation c.1072C>T (p.Arg358X) in the EDAR gene. A review of the literature with regard to other cases presenting the same mutation has been carried out and discussed.

Somatic and germline duplications or activating mutations of AKT3 have been reported in patients with hemimegalencephaly and megalencephaly. We performed array comparative genomic hybridization on brain tissue and blood in 16 consecutive patients with symptomatic epilepsy due to focal or multilobar malformations of cortical development who underwent surgical treatment of epilepsy. One patient with infantile spasms and a dysplastic left frontal lobe harboured a somatic trisomy of the 1q21.1-q44 chromosomal region, encompassing the AKT3 gene, in the dysplastic brain tissue but not in blood and saliva. Histopathology revealed severe cortical dyslamination, a thin cortex in the premotor area with microgyri and microsulci, immature neurons with disoriented dendrites and areas of cortical heterotopia in the sub-cortical white matter. These cytoarchitectural changes are close to those defining type Ib focal cortical dysplasia. Immunohistochemistry in brain specimens showed hyperactivation of the PI3K/AKT/mTOR pathway. These findings indicate that AKT3 upregulation may cause focal malformations of cortical development. There appears to be an etiologic continuum between hemimegalencephaly and focal cortical dysplastic lesions. The extent of brain malformations due to AKT3 upregulation may be related to the embryonic stage when the post-zygotic gene alteration occurs.

Identification of the genetic defect underlying early-onset diabetes is important for determining the specific diabetes subtype, which would then permit appropriate treatment and accurate assessment of recurrence risk in offspring. Given the extensive genetic and clinical heterogeneity of the disease, high-throughput sequencing might provide additional diagnostic potential when Sanger sequencing is ineffective. Our aim was to develop a targeted next-generation assay able to detect mutations in several genes involved in glucose metabolism. All 13 known MODY genes, genes identified from a genome-wide linkage study or genome-wide association studies as increasing the risk of type 2 diabetes and genes causing diabetes in animal models, were included in the custom panel. We selected a total of 102 genes by performing a targeting re-sequencing in 30 patients negative for mutations in the GCK, HNF1α, HNF4α, HNF1β and IPF1 genes at the Sanger sequencing analysis. Previously unidentified variants in the RFX6 gene were found in three patients and in two of them we also detected rare variants in WFS1 and ABCC8 genes. All patients showed a good therapeutic response to dipeptidyl peptidase-4 (DPP4) inhibitors. Our study reveals that next-generation sequencing provides a highly sensitive method for identification of variants in new causative genes of diabetes. This approach may help in understanding the molecular etiology of diabetes and in providing more personalized treatment for each genetic subtype.

Ectodermal dysplasias (EDs) are a group of genetic disorders characterized by the abnormal development of the ectodermal-derived structures. X-linked hypohidrotic ectodermal dysplasia, resulting from mutations in ED1 gene, is the most common form. The main purpose of this study was to characterize the phenotype spectrum in 45 males harboring ED1 mutations. The study showed that in addition to the involvement of the major ectodermal tissues, the majority of patients also have alterations of several minor ectodermal-derived structures. Characterizing the clinical spectrum resulting from ED1 gene mutations improves diagnosis and can direct clinical care.

We report four cases of subjects with phenotypic abnormalities and mental retardation associated with apparently balanced translocations, two inherited and two de novo, which showed, by molecular analysis, a hidden complexity. All the cases have been analyzed with different molecular techniques, including array-CGH, and in two of them the translocation breakpoints have been defined at the level of base pairs via studies in somatic hybrids containing single derivative chromosomes. We demonstrated that all the translocations were in fact complex rearrangements and that an imbalance was present in three of them, thus accounting for the phenotypic abnormalities. In one case, a Prader-Willi subject, we were not able to determine the molecular cause of his phenotype. This study, while confirming previous data showing unexpected complexity in translocations, further underscores the need for molecular investigations before taking for granted an apparently simple cytogenetic interpretation.

We report on a 5p- azoospermic male not showing the clinical features diagnostic for the cri-du-chat syndrome but for a breathy, raspy voice. The 5p deletion breakpoint, determined by fluorescent in situ hybridization (FISH) analysis with BAC clones, maps 8.5 Mb far from the short arm telomere in 5p15.31. Genotype/phenotype correlations in this subject, including his neuropsychological assessment, led us to define that the gene for the cat-like cry and one gene responsible for mild mental retardation with speech delay map both in the distal 8.5 Mb of chromosome 5 short arm.

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART. © 2015 American Society of andrology and European Academy of andrology.

Duplications in the ~2 Mb desert region upstream of SOX9 at 17q24.3 may result in familial 46,XX disorders of sex development (DSD) without any effects on the XY background. A balanced translocation with its breakpoint falling within the same region has also been described in one XX DSD subject. We analyzed, by conventional and molecular cytogenetics, 19 novel SRY-negative unrelated 46,XX subjects both familial and sporadic, with isolated DSD. One of them had a de novo reciprocal t(11;17) translocation. Two cases carried partially overlapping 17q24.3 duplications ~500 kb upstream of SOX9, both inherited from their normal fathers. Breakpoints cloning showed that both duplications were in tandem, whereas the 17q in the reciprocal translocation was broken at ~800 kb upstream of SOX9, which is not only close to a previously described 46,XX DSD translocation, but also to translocations without any effects on the gonadal development. A further XX male, ascertained because of intellectual disability, carried a de novo cryptic duplication at Xq27.1, involving SOX3. CNVs involving SOX3 or its flanking regions have been reported in four XX DSD subjects. Collectively in our cohort of 19 novel cases of SRY-negative 46,XX DSD, the duplications upstream of SOX9 account for ~10.5% of the cases, and are responsible for the disease phenotype, even when inherited from a normal father. Translocations interrupting this region may also affect the gonadal development, possibly depending on the chromatin context of the recipient chromosome. SOX3 duplications may substitute SRY in some XX subjects.

The t(4;8)(p16;p23) translocation, in either the balanced form or the unbalanced form, has been reported several times. Taking into consideration the fact that this translocation may be undetected in routine cytogenetics, we find that it may be the most frequent translocation after t(11q;22q), which is the most common reciprocal translocation in humans. Case subjects with der(4) have the Wolf-Hirschhorn syndrome, whereas case subjects with der(8) show a milder spectrum of dysmorphic features. Two pairs of the many olfactory receptor (OR)–gene clusters are located close to each other, on both 4p16 and 8p23. Previously, we demonstrated that an inversion polymorphism of the OR region at 8p23 plays a crucial role in the generation of chromosomal imbalances through unusual meiotic exchanges. These findings prompted us to investigate whether OR-related inversion polymorphisms at 4p16 and 8p23 might also be involved in the origin of the t(4;8)(p16;p23) translocation. In seven case subjects (five of whom both represented de novo cases and were of maternal origin), including individuals with unbalanced and balanced translocations, we demonstrated that the breakpoints fell within the 4p and 8p OR-gene clusters. FISH experiments with appropriate bacterial-artificial-chromosome probes detected heterozygous submicroscopic inversions of both 4p and 8p regions in all the five mothers of the de novo case subjects. Heterozygous inversions on 4p16 and 8p23 were detected in 12.5% and 26% of control subjects, respectively, whereas 2.5% of them were scored as doubly heterozygous. These novel data emphasize the importance of segmental duplications and large-scale genomic polymorphisms in the evolution and pathology of the human genome.

Connexin36 (Cx36) is a new connexin that was recently cloned in mouse, rat and human. It is highly expressed in neurons of the CNS. To gain insight into the transcriptional regulation of this gene, we have cloned the genomic region containing the entire mCx36 gene and sequenced about 7.6 kb around the coding region. The computer analysis of this sequence was helpful in defining putative regulative sequences. Using both 5′-RACE and RNAse protection assay, we have mapped the transcription starting site commonly used in both adult olfactory bulb and brain, in position −479 from the ATG. By 3′-RACE, we defined the polyadenylation site used that is located 1436 nt downstream the stop codon. The expected transcript is 2875 nt long and is consistent with the 2.9 kb transcript found in the same tissues by Northern blot. Finally, we have mapped mCx36 on chromosome 2 in the position F3 in a region that is synthenic to human chromosome 15q14, where the human Cx36 gene has been recently mapped.

An unusual Y chromosome with a primary constriction inside the long-arm heterochromatin was found in the amniocytes of a 38-year-old woman. The same Y chromosome was found in her husband and brother-in-law, thus proving that it was already present in the father. FISH with alphoid DNA showed hybridization signals at the usual position of the Y centromere but not at the primary constriction. Centromere proteins (CENP)-A, CENP-C, and CENP-E could not be detected at the site of the canonic centromere but were present at the new constriction, whereas CENP-B was not detected on this Y chromosome. Experiments with 82 Y-specific loci distributed throughout the chromosome confirmed that no gross deletion or rearrangement had taken place, and that the Y chromosome belonged to a haplogroup whose members have a mean alphoid array of 770 kb (range 430-1,600 kb), whereas that of this case was approximately 250 kb. Thus, this Y chromosome appeared to be deleted for part of the alphoid DNA. It seems likely that this deletion was responsible for the silencing of the normal centromere and that the activation of the neocentromere prevented the loss of this chromosome. Alternatively, neocentromere activation could have occurred first and stimulated inactivation of the normal centromere by partial deletion. Whatever the mechanism, the presence of this chromosome in three generations demonstrates that it functions sufficiently well in mitosis for male sex determination and fertility and that neocentromeres can be transmitted normally at meiosis.

Glial cells play fundamental roles in neurogenesis. In addition, defective or abnormal gliogenesis is associated with severe diseases. To understand the molecular basis of such diseases, it is crucial to identify the genes promoting normal gliogenesis. Here we identify GCMB, which encodes a human protein homologous to the fly glial promoting factor glial cell deficient/glial cell missing (glide/gcm).

The human X chromosome is known to contain several disease genes yet to be cloned. In the course of a project aimed at the construction of a transcription map of the Xp22 region, we fully characterized a novel cDNA, Cxorf5 (HGMW-approved symbol, alias 71-7A), previously mapped to this region but for which no sequence information was available. We isolated and sequenced the full-length transcript, which encodes a predicted protein of unknown function containing a large number of coiled-coild domains, typically presented in a variety of different molecules, from fibrous proteins to transcription factors. We showed that the Cxorf5 cDNA is ubiquitously expressed, undergoes alternative splicing, and escapes X inactivation. Furthermore, we precisely mapped two additional Cxorf5-related loci on the Y chromosome and on chromosome 5. By virtue of its mapping assignment to the Xp22 region, Cxorf5 represents a candidate gene for at least four human diseases, namely spondyloepiphiseal dysplasia late, oral-facial-digital syndrome type 1, craniofrontonasal syndrome, and a nonsyndromic sensorineural deafness.

A novel INDEL mutation in theEDA gene resulting in a distinctX- linked hypohidroticectoder mal dysplasia phenotypein an Italian familyEditorX-Linked Hypohidrotic Ectodermal Dysplasia (XL-HED; MIM305100) is characterized by hypodontia, misshaped teeth, hypo-hidrosis, sparse hair, peculiar facial features,1,2and occurs in lessthan 1 in every 100.000 individuals.1XL-HED is caused bymutations in the Ectodysplasin-A (EDA) gene located at Xq12-q13 with more than 100 causative mutations reported todate.1,3,4The identification of disease-causing mutations con-firms the diagnosis, however, does not automatically imply agenotype–phenotype correlation.

Identification of a novelframeshift mutation in theEDAR gene causing autosomaldominant hypohidroticectoder mal dysplasiaEditorThe ectodermal dysplasias (EDs) are a group of inherited disor-ders affecting ectodermal-derived tissues, including the hair,nails, teeth, skin and sweat glands.1,2Hypohidrotic ectodermaldysplasia (HED) represents one of the major types of ED andresults from mutations in the EDA (MIM *300451), EDAR(MIM *604095) EDARADD (MIM *606603) and TRAF6 (MIM*602355) genes. The inheritance of HED can be X-linked(XLHED; MIM#305100) or autosomal either dominant(MIM#129490) or recessive (MIM#305100).3,4No definitegenotype–phenotype correlations have been established to date.However, recently identified EDAR mutations demonstrate thatpathogenic variants result in variable phenotypes with mild-to-severe clinical manifestations.5We report a novel mutation in the EDAR gene in an Italianfamily with autosomal dominant HED that supports emergingevidence for a genoytype–phenotype correlation.5The clinical,genetic and functional studies were conducted according to theHelsinki declaration and written informed consent was obtainedfrom all participating family members; including consent topublish data and clinical images. Three members of the familywere available for detailed clinical investigation (V:5; IV:7; IV:5),and four underwent molecular genetic analysis (V:5; IV:7; IV:5;III:2)

Morquio A syndrome (MPS IVA) is a systemic lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), encoded by the GALNS gene. We studied 37 MPS IV A patients and defined genotype-phenotype correlations based on clinical data, biochemical assays, molecular analyses, and in silico structural analyses of associated mutations. We found that standard sequencing procedures, albeit identifying 14 novel small GALNS genetic lesions, failed to characterize the second disease-causing mutation in the 16% of the patients' cohort. To address this drawback and uncover potential gross GALNS rearrangements, we developed molecular procedures (CNV [copy-number variation] assays, QF-PCRs [quantitative fluorescent-PCRs]), endorsed by CGH-arrays. Using this approach, we characterized two new large deletions and their corresponding breakpoints. Both deletions were heterozygous and included the first exon of the PIEZO1 gene, which is associated with dehydrated hereditary stomatocitosis, an autosomal-dominant syndrome. In addition, we characterized the new GALNS intronic lesion c.245-11C>G causing m-RNA defects, although identified outside the GT/AG splice pair. We estimated the occurrence of the disease in the Italian population to be approximately 1:300,000 live births and defined a molecular testing algorithm designed to help diagnosing MPS IVA and foreseeing disease progression.

Background Hamartomatous polyposis syndromes (HPS) are inherited conditions associated with high cancer risk. They include the Peutz-Jeghers and the PTEN hamartoma tumor syndromes, which are caused by mutations in the LKB1 and PTEN genes, respectively. Estimation of cancer risk is crucial in order to optimize surveillance, but no prognostic markers are currently available for these conditions. Our study relies on a ‘signal transduction’ hypothesis based on the crosstalk between LKB1/AMPK and PI3K/PTEN/Akt signaling at the level of the tumor suppressor protein FoxO3A. Interestingly, the FOXO3A rs2802292 G-allele was shown to be associated with longevity, reduced risk of aging-related diseases and increased expression of FoxO3A mRNA. Methods We typed rs2802292 in 150 HPS unrelated patients and characterized the expression of FoxO3A by quantitative PCR and immunoblot analysis in human intestinal cell lines. Results We found a significantly higher risk for malignancies in females and TT genotype carriers compared to patients having at least one G-allele. Subgroup analysis for each HPS syndrome revealed a G-allele-associated beneficial effect on cancer risk occurring mainly in males. Molecular characterization of human intestinal cell lines showed that the G-allele significantly correlated with increased basal expression of FoxO3A mRNA and protein. Conclusion Our results suggest an inverse correlation between the protective allele (G) copy number and cancer risk, and might be useful to optimize surveillance in HPS patients. Further investigations are needed to confirm our hypothesis and to ascertain whether differences in therapeutic response exist across genotypes.

Cross-species comparison is an effective tool used to identify genes and study their function in both normal and pathological conditions. We have applied the power of Drosophila genetics to the vast resource of human cDNAs represented in the expressed sequence tag (EST) database (dbEST) to identify novel human genes of high biological interest. Sixty-six human cDNAs showing significant homology to genes causing Drosophila mutant phenotypes were identified by screening dbEST using the "text string' option, and their map position was determined using both fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Comparison between these genes and their putative partners in Drosophila may provide important insights into their function in mammals. Furthermore, integration of these genes into the transcription map of the human genome contributes to the positional candidate approach for disease gene identification.

Interstitial chromosome 15q11-q13 duplications are associated with developmental delay, behavioral problems and additional manifestations, including epilepsy. In most affected individuals the duplicated chromosome is maternally derived, whereas paternal inheritance is more often associated with a normal phenotype. Seizures have not been described in patients with paternal dup 15q11-q13. We describe a family with five individuals in three generations with a paternally-inherited 15q11-q13 duplication, four of whom exhibited abnormal phenotypic characteristics, including seizures. The 18-year-old female proband presented with moderate intellectual disability, obesity, and epilepsy. Her brother manifested learning disability and behavioral problems. They both inherited the 15q11-q13 dup from their father who had a normal phenotype. Their paternal uncle and grandfather also had the duplication and were reported to have had seizures. Array-CGH and MLPA analyses showed that the duplication included the TUBGCP5, CYFIP1, MKRN3, MAGEL2, NDN, SNRPN, UBE3A, ATP10A, GABRB3, GABRA5, GABRG3, and OCA2 genes. This report provides evidence for intrafamilial phenotypic variability of paternal dup 15q11-q13, ranging from normal to intellectual disability and seizures, and potentially expanding the phenotype of paternal 15q11-q13 interstitial duplications.