Split Hand-Foot and Deafness in a Patient with 7q21.13-q21.3 Deletion Not Including the DLX5/6 Genes.

Split Hand-Foot Malformation (SHFM) is a congenital limb defect characterized by a median cleft of the hands and/or feet due to the absence/hypoplasia of the central rays. It may occur as part of a syndromic condition or as an isolated malformation. The most common of the six genetic loci identified for this condition is correlated to SHFM1 and maps in the 7q21q22 region. SHFM1 is characterized by autosomal dominant transmission, incomplete penetrance and variable expressivity. Associated features often include hearing loss, intellectual disability/developmental delay and craniofacial abnormalities. Disruption of the DLX5/DLX6 genes, mapping within the SHFM1 locus, is now known to be responsible for the phenotype. Through SNP array, we analyzed a patient affected by SHFM1 associated with deafness and an abnormality of the inner ear (incomplete partition type I); we identified a deletion in 7q21, not involving the DLX5/6 genes, but including exons 15 and 17 of DYNC1I1, known to act as exonic enhancers (eExons) of the DLX5/6 genes. We further demonstrated the role of DYNC1I1 eExons in regulating DLX5/6 expression by means of showing a reduced expression of the DLX5/6 genes through RT-PCR in a patient-derived lymphoblastoid cell line. Furthermore, our data and a review of published cases do not support the hypothesis that DLX5/6 are imprinted in humans. This work is an example of how the disruption of regulatory elements can be responsible for congenital malformations.

2p25.3 microduplications involving MYT1L: further phenotypic characterization through an assessment of 16 new cases and a literature review.

Microduplications involving the MYT1L gene have mostly been described in series of patients with isolated schizophrenia. However, few reports have been published, and the phenotype has still not been well characterized. We sought to further characterize the phenotypic spectrum of this condition by describing the clinical features of patients with a pure 2p25.3 microduplication that includes all or part of MYT1L. We assessed 16 new patients with pure 2p25.3 microduplications recruited through a French national collaboration (n = 15) and the DECIPHER database (n = 1). We also reviewed 27 patients reported in the literature. For each case, we recorded clinical data, the microduplication size, and the inheritance pattern. The clinical features were variable and included developmental and speech delays (33%), autism spectrum disorder (ASD, 23%), mild-to-moderate intellectual disability (ID, 21%), schizophrenia (23%), or behavioral disorders (16%). Eleven patients did not have an obvious neuropsychiatric disorder. The microduplications ranged from 62.4 kb to 3.8 Mb in size and led to duplication of all or part of MYT1L; seven of these duplications were intragenic. The inheritance pattern was available for 18 patients: the microduplication was inherited in 13 cases, and all parents but one had normal phenotype. Our comprehensive review and expansion of the phenotypic spectrum associated with 2p25.3 microduplications involving MYT1L should help clinicians to better assess, counsel and manage affected individuals. MYT1L microduplications are characterized by a spectrum of neuropsychiatric phenotypes with incomplete penetrance and variable expressivity, which are probably due to as-yet unknown genetic and nongenetic modifiers.

3'UTR Deletion of NONO Leads to Corpus Callosum Anomaly, Left Ventricular Non-Compaction and Ebstein's Anomaly in a Male Fetus.

NONO (Non-Pou Domain-Containing Octamer-Binding Protein) gene maps on chromosome Xq13.1 and hemizygous loss-of-function nucleotide variants are associated with an emerging syndromic form of intellectual developmental disorder (MRXS34; MIM #300967), characterized by developmental delay, intellectual disability, poor language, dysmorphic facial features, and microcephaly. Structural brain malformation, such as corpus callosum and cerebellar abnormalities, and heart defects, in particular left ventricular non-compaction (LVNC), represent the most recurrent congenital malformations, recorded both in about 80% of patients, and can be considered the distinctive imaging findings of this disorder. We present on a further case of NONO-related disease; prenatally diagnosed in a fetus with complete corpus callosum agenesis; absence of septum pellucidum; pericallosal artery; LVNC and Ebstein's anomaly. A high-resolution microarray analysis demonstrated the presence of a deletion affecting the NONO 3'UTR; leading to a marked hypoexpression of the gene and the complete absence of the protein in cultured amniocytes. This case expands the mutational spectrum of MRXS34, advises to evaluate NONO variants in pre- and postnatal diagnosis of subjects affected by LVNC and other heart defects, especially if associated with corpus callosum anomalies and confirm that CNVs (Copy Number Variants) represent a non-negligible cause of Mendelian disorders.

Chromosomal Microarray Analysis in Fetuses Detected with Isolated Cardiovascular Malformation: A Multicenter Study, Systematic Review of the Literature and Meta-Analysis.

Cardiovascular malformations (CVM) represent the most common structural anomalies, occurring in 0.7% of live births. The CVM prenatal suspicion should prompt an accurate investigation with fetal echocardiography and the assessment through genetic counseling and testing. In particular, chromosomal microarray analysis (CMA) allows the identification of copy number variations. We performed a systematic review and meta-analysis of the literature, studying the incremental diagnostic yield of CMA in fetal isolated CVM, scoring yields for each category of heart disease, with the aim of guiding genetic counseling and prenatal management. At the same time, we report 59 fetuses with isolated CVM with normal karyotype who underwent CMA. The incremental CMA diagnostic yield in fetuses with isolated CVM was 5.79% (CI 5.54-6.04), with conotruncal malformations showing the higher detection rate (15.93%). The yields for ventricular septal defects and aberrant right subclavian artery were the lowest (2.64% and 0.66%). Other CVM ranged from 4.42% to 6.67%. In the retrospective cohort, the diagnostic yield was consistent with literature data, with an overall CMA diagnostic yield of 3.38%. CMA in the prenatal setting was confirmed as a valuable tool for investigating the causes of fetal cardiovascular malformations.

A novel complex genomic rearrangement affecting the KCNJ2 regulatory region causes a variant of Cooks syndrome.

Cooks syndrome (CS) is an ultrarare limb malformation due to in tandem microduplications involving KCNJ2 and extending to the 5' regulatory element of SOX9. To date, six CS families were resolved at the molecular level. Subsequent studies explored the evolutionary and pathological complexities of the SOX9-KCNJ2/Sox9-Kcnj2 locus, and suggested a key role for the formation of novel topologically associating domain (TAD) by inter-TAD duplications in causing CS. Here, we report a unique case of CS associated with a de novo 1;17 translocation affecting the KCNJ2 locus. On chromosome 17, the breakpoint mapped between KCNJ16 and KCNJ2, and combined with a ~ 5 kb deletion in the 5' of KCNJ2. Based on available capture Hi-C data, the breakpoint on chromosome 17 separated KCNJ2 from a putative enhancer. Gene expression analysis demonstrated downregulation of KCNJ2 in both patient's blood cells and cultured skin fibroblasts. Our findings suggest that a complex rearrangement falling in the 5' of KCNJ2 may mimic the developmental consequences of in tandem duplications affecting the SOX9-KCNJ2/Sox9-Kcnj2 locus. This finding adds weight to the notion of an intricate role of gene regulatory regions and, presumably, the related three-dimensional chromatin structure in normal and abnormal human morphology.

De Novo Inverted Duplication Deletion of 4p in a 14-Week-Old Male Fetus Aborted Due to Multiple Anomalies.

Inverted duplications deletions are rare, complex, and nonrecurrent chromosomal rearrangements associated with a variable phenotype. In this case report, we described the phenotype and genotype of a 14-week-old male fetus, who was aborted after discovery of multiple anomalies (septal cystic hygroma, open abdominal wall, and a nonidentifiable lower limb). At autopsy, fluorescence in situ hybridization and array comparative genomic hybridization identified an inverted duplication with terminal deletion of 4p [46,XY,der(4)del(p16.3)dup(4)(p15.2p16.3)]. Only five genotypically similar cases have been reported, and we hope our case contribution will add meaningful to the body of knowledge.

Copy number variation analysis implicates novel pathways in patients with oculo-auriculo-vertebral-spectrum and congenital heart defects.

Oculo-auriculo-vertebral spectrum (OAVS) is a developmental disorder of craniofacial morphogenesis. Its etiology is unclear, but assumed to be complex and heterogeneous, with contribution of both genetic and environmental factors. We assessed the occurrence of copy number variants (CNVs) in a cohort of 19 unrelated OAVS individuals with congenital heart defect. Chromosomal microarray analysis identified pathogenic CNVs in 2/19 (10.5%) individuals, and CNVs classified as variants of uncertain significance in 7/19 (36.9%) individuals. Remarkably, two subjects had small intragenic CNVs involving DACH1 and DACH2, two paralogs coding for key components of the PAX-SIX-EYA-DACH network, a transcriptional regulatory pathway controlling developmental processes relevant to OAVS and causally associated with syndromes characterized by craniofacial involvement. Moreover, a third patient showed a large duplication encompassing DMBX1/OTX3, encoding a transcriptional repressor of OTX2, another transcription factor functionally connected to the DACH-EYA-PAX network. Among the other relevant CNVs, a deletion encompassing HSD17B6, a gene connected with the retinoic acid signaling pathway, whose dysregulation has been implicated in craniofacial malformations, was also identified. Our findings suggest that CNVs affecting gene dosage likely contribute to the genetic heterogeneity of OAVS, and implicate the PAX-SIX-EYA-DACH network as novel pathway involved in the etiology of this developmental trait.

12q21 deletion syndrome: Narrowing the critical region down to 1.6 Mb including SYT1 and PPP1R12A.

Deletions in the 12q21 region are rare and non-recurrent CNVs. To date, only 11 patients with deletions in this region have been reported in the literature. These patients most often presented with syndromic intellectual deficiency, ventriculomegaly or hydrocephalus, ectodermal abnormalities, growth retardation and renal and cardiac malformations, suggesting a recognizable microdeletion syndrome. We report three new patients with overlapping deletions of the 12q21 region, including the smallest deletion reported to date and the first case characterized by array CGH during pregnancy. We describe specific clinical findings and shared facial features as developmental delay, ectodermal abnormalities, ventriculomegaly or hydrocephalus, axial hypotonia or spastic diplegia, growth retardation, heart defect, hydronephrosis, ureteral reflux or horseshoe kidney, large thorax or pectus excavatum, syndactyly of 2-3 toes, pterygium coli or excess nuchal skin, large anterior fontanel, low set ears, prominent forehead, short-upturned nose with nostril hypoplasia, microretrognathia and hypertelorism. These new patients and a comprehensive review of the literature allow us to define a minimum critical region spanning 1.6 Mb in 12q21. By screening the critical region using prediction tools, we identified two candidate genes: SYT1and PPP1R12A.

A novel mosaic 1q32.1 microduplication identified through Chromosome Microarray Analysis: narrowing the smallest critical region including KDM5B gene found associated with neurodevelopmetal disorders.

Microduplications involving 1q32.1 chromosomal region have been rarely reported in literature. Patients with these microduplications suffer from intellectual disability, developmental delay and a number of dysmorphic features, although no clear karyotype/phenotype correlation has yet been determined. In this case report we describe two monochorionic-diamniotic twins with intellectual disability, abnormality of coordination and dysmorphic features associated with a de novo 280 kb mosaic microduplication of 1q32.1 chromosomal region, identified using a Chromosome Microarray Analysis (CMA) and confirmed by quantitative PCR analysis. The duplicated region encompassed entirely three OMIM genes KDM5B (*605393), KLHL12 (*614522), RABIF (*603417) and involved partially SYT2 (*600104). This unique case report allows to redefine the critical 1q32.1 microduplicated region implicated in the ethiopathogenesis of intellectual disability and developmental delay. Furthermore, it suggests that KDM5B gene can have a pivotal role in the development of neurodevelopmental disorders through its demethylase activity.

Progenitor/Stem Cell Markers in Brain Adjacent to Glioblastoma: GD3 Ganglioside and NG2 Proteoglycan Expression.

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Predictive diagnostic value for the clinical features accompanying intellectual disability in children with pathogenic copy number variations: a multivariate analysis.

BACKGROUND: Array comparative genomic hybridization (a-CGH) has become the first-tier investigation in patients with unexplained developmental delay/intellectual disability (DD/ID). Although the costs are progressively decreasing, a-CGH is still an expensive and labour-intensive technique: for this reason a definition of the categories of patients that can benefit the most of the analysis is needed. Aim of the study was to retrospectively analyze the clinical features of children with DD/ID attending the outpatient clinic of the Mother & Child Department of the University Hospital of Modena subjected to a-CGH, to verify by uni- and multivariate analysis the independent predictors of pathogenic CNVs. METHODS: 116 patients were included in the study. Data relative to the CNVs and to the patients' clinical features were analyzed for genotype/phenotype correlations. RESULTS AND CONCLUSIONS: 27 patients (23.3%) presented pathogenic CNVs (21 deletions, 3 duplications and 3 cases with both duplications and deletions). Univariate analysis showed a significant association of the pathogenic CNVs with the early onset of symptoms (before 1 yr of age) and the presence of malformations and dysmorphisms. Logistic regression analysis showed a significant independent predictive value for diagnosing a pathogenic CNV for malformations (P = 0.002) and dysmorphisms (P = 0.023), suggesting that those features should address a-CGH analysis as a high-priority test for diagnosis.

17p13.1 microdeletion: genetic and clinical findings in a new patient with epilepsy and comparison with literature.

Array comparative genomic hybridization is now a powerful tool to investigate patients with multiple congenital abnormalities and intellectual/motor impairment, and genomic imbalances are identified in a growing number of children with intellectual disability. Deletions in the 17p13.1 region have been reported in patients with dysmorphic features and developmental delay but a consistent phenotype has yet to emerge. Here, we report on the diagnosis of a 17p13.1 microdeletion of 829 kb in an 8-year-old girl presenting with profound cognitive disability, psychomotor delay, facial dysmorphisms, and refractory epilepsy. This deletion comprises 44 genes, including 8 OMIM morbid genes. We discuss genetic, clinical, and epileptic features comparing our patient with those previously reported in the literature.

Ebstein anomaly: Genetic heterogeneity and association with microdeletions 1p36 and 8p23.1.

Ebstein anomaly is an uncommon congenital heart defect (CHD), characterized by downward displacement of the tricuspid valve into the right ventricle. To uncover the genetic associations with Ebstein anomaly, we have searched chromosomal imbalances using standard cytogenetic and array-CGH analysis, and single gene conditions associated with syndromic Ebstein anomaly (with extracardiac anomalies), and screened GATA4 and NKX2.5 mutations in nonsyndromic patients (without extracardiac anomalies). Between January 1997 and September 2009, 44 consecutive patients with Ebstein anomaly were evaluated in two centers of Pediatric Cardiology. Ebstein anomaly was syndromic in 12 (27%) patients, and nonsyndromic in 32 (73%). A recognizable syndrome or complex was diagnosed by clinical criteria in seven patients. In one syndromic patient an 18q deletion was diagnosed by standard cytogenetic analysis. Array-CGH analysis performed in 10 of the 12 syndromic patients detected an interstitial deletion of about 4 Mb at 8p23.1 in one patient, and a deletion 1pter > 1p36.32/dup Xpter- > Xp22.32 in another patient. In the 28 of 32 nonsyndromic patients who underwent molecular testing, no mutation in GATA4 and NKX2.5 genes were detected. We conclude that Ebstein anomaly is a genetically heterogeneous defect, and that deletion 1p36 and deletion 8p23.1 are the most frequent chromosomal imbalances associated with Ebstein anomaly. Candidate genes include the GATA4 gene (in patients with del 8p23.1), NKX2.5 (based on published patients with isolated Ebstein anomaly) and a hypothetical gene in patients with del 1p36).

X chromosome monosomy restricted to the left ventricle is not a major cause of isolated hypoplastic left heart.

Hypoplastic left heart sequence (HLHS) encompasses obstructive lesions of the left side of the heart accompanied by varying degrees of underdevelopment of the mitral valve, either atresia or stenosis, left ventricle, aortic valve, and ascending aorta. HLHS has an incidence of 0.016-0.036% live births and can occur as an isolated defect or several different chromosome abnormalities. In particular, about 20% of patients with monosomy X, have HLHS, suggesting a relationship between this form of aneuploidy and this congenital heart defect (CHD). Somatic mutations restricted to the cardiac affected structure have been considered a mechanism of CHD. The aim of this study was to evaluate if monosomy X restricted to the left ventricle causes isolated HLHS. Formalin-fixed, paraffin-embedded cardiac tissue obtained from 19 patients with HLHS (10 males and 9 females) without extra cardiac anomalies and with a normal constitutional karyotype, were investigated by FISH analysis, using X/Y/18 centromeric probes. The results of this analysis were compared with those obtained by examining the heart specimens of 15 chromosomally normal pediatric patients affected by either restrictive or dilated cardiomyopathy, which were used as negative controls. Mosaic monosomy X was detected in the cardiac tissue nuclei of both groups, with similar frequencies (6-16% and 12-16%, respectively), suggesting that chromosome X monosomy is not rare in this tissue, but is not a major cause of isolated HLHS.

3q29 Microdeletion: a mental retardation disorder unassociated with a recognizable phenotype in two mother-daughter pairs.

The 3q29 microdeletion syndrome (del 3q29) is a novel genomic disorder identified after the introduction of microarray-based technology. The phenotype of the reported patients is variable, including mental retardation and subtle facial anomalies. We report on two mother-daughter pairs, heterozygous for 3q29, and review clinical features of all known affected individuals. Del 3q29 syndrome is associated with nonspecific clinical features, including mild-to-moderate developmental delay, microcephaly, and mild facial dysmorphisms such as short philtrum and high nasal bridge. Facial anomalies were nonoverlapping and nondistinct, also within each mother-daughter pair. Parental transmission of del 3q29 could be more frequent than previously considered. Malformations are rare, occurring only in single subjects. The phenotypic diversity of affected patients and the lack of distinct dysmorphisms suggest that this disorder cannot be recognized on clinical ground alone. Del 3q29 should be searched in subjects with unexplained mild/moderate mental retardation, microcephaly, and minor nonspecific facial anomalies.

Association of syndromic mental retardation with an Xq12q13.1 duplication encompassing the oligophrenin 1 gene.

OPHN1 mutations cause a syndromic form of mental retardation (MR) characterized by cerebellar hypoplasia, early hypotonia, motor and speech delay, with occasional seizures and strabismus. Here we report on a familial chromosome duplication spanning about 800 Kb of Xq12q13.1, associated with MR and a distinctive phenotype in the affected male, but not in his heterozygous mother. The parents were healthy and non-consanguineous with a history of three pregnancies. The first resulted in the birth of a boy with MR, motor impairment and seizures. The second pregnancy was terminated because of trisomy 18. At the time of the third, the first affected boy was analyzed by array-CGH, which revealed a 800 Kb duplication at Xq12q13.1, encompassing three genes, including OPHN1. This mutation was inherited from his healthy mother and was not present in any of the three maternal brothers. To our knowledge this is the first report of a clinical phenotype associated with duplication of Xq12q13.