Novel promoters and coding first exons in DLG2 linked to developmental disorders and intellectual disability.

BACKGROUND: Tissue-specific integrative omics has the potential to reveal new genic elements important for developmental disorders. METHODS: Two pediatric patients with global developmental delay and intellectual disability phenotype underwent array-CGH genetic testing, both showing a partial deletion of the DLG2 gene. From independent human and murine omics datasets, we combined copy number variations, histone modifications, developmental tissue-specific regulation, and protein data to explore the molecular mechanism at play. RESULTS: Integrating genomics, transcriptomics, and epigenomics data, we describe two novel DLG2 promoters and coding first exons expressed in human fetal brain. Their murine conservation and protein-level evidence allowed us to produce new DLG2 gene models for human and mouse. These new genic elements are deleted in 90% of 29 patients (public and in-house) showing partial deletion of the DLG2 gene. The patients' clinical characteristics expand the neurodevelopmental phenotypic spectrum linked to DLG2 gene disruption to cognitive and behavioral categories. CONCLUSIONS: While protein-coding genes are regarded as well known, our work shows that integration of multiple omics datasets can unveil novel coding elements. From a clinical perspective, our work demonstrates that two new DLG2 promoters and exons are crucial for the neurodevelopmental phenotypes associated with this gene. In addition, our work brings evidence for the lack of cross-annotation in human versus mouse reference genomes and nucleotide versus protein databases.

Common PHOX2B poly-alanine contractions impair RET gene transcription, predisposing to Hirschsprung disease.

HSCR is a congenital disorder of the enteric nervous system, characterized by the absence of neurons along a variable length of the gut resulting from loss-of-function RET mutations. Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by impaired response to hypercapnia and hypoxemia caused by heterozygous mutations of the PHOX2B gene, mostly polyalanine (polyA) expansions but also missense, nonsense, and frameshift mutations, while polyA contractions are common in the population and believed neutral. HSCR associated CCHS can present in patients carrying PHOX2B mutations. Indeed, RET expression is orchestrated by different transcriptional factors among which PHOX2B, thus suggesting its possible role in HSCR pathogenesis. Following the observation of HSCR patients carrying in frame trinucleotide deletions within the polyalanine stretch in exon 3 (polyA contractions), we have verified the hypothesis that these PHOX2B variants do reduce its transcriptional activity, likely resulting in a down-regulation of RET expression and, consequently, favouring the development of the HSCR phenotype. Using proper reporter constructs, we show here that the in vitro transactivation of the RET promoter by different HSCR-associated PHOX2B polyA variants has resulted significantly lower compared to the effect of PHOX2B wild type protein. In particular, polyA contractions do induce a reduced transactivation of the RET promoter, milder compared to the severe polyA expansions associated with CCHS+HSCR, and correlated with the length of the deleted trait, with a more pronounced effect when contractions are larger.

Neuroimaging findings in Mowat-Wilson syndrome: a study of 54 patients.

PURPOSE: Mowat-Wilson syndrome (MWS) is a genetic disease characterized by distinctive facial features, moderate to severe intellectual disability, and congenital malformations, including Hirschsprung disease, genital and eye anomalies, and congenital heart defects, caused by haploinsufficiency of the ZEB2 gene. To date, no characteristic pattern of brain dysmorphology in MWS has been defined. METHODS: Through brain magnetic resonance imaging (MRI) analysis, we delineated a neuroimaging phenotype in 54 MWS patients with a proven ZEB2 defect, compared it with the features identified in a thorough review of published cases, and evaluated genotype-phenotype correlations. RESULTS: Ninety-six percent of patients had abnormal MRI results. The most common features were anomalies of corpus callosum (79.6% of cases), hippocampal abnormalities (77.8%), enlargement of cerebral ventricles (68.5%), and white matter abnormalities (reduction of thickness 40.7%, localized signal alterations 22.2%). Other consistent findings were large basal ganglia, cortical, and cerebellar malformations. Most features were underrepresented in the literature. We also found ZEB2 variations leading to synthesis of a defective protein to be favorable for psychomotor development and some epilepsy features but also associated with corpus callosum agenesis. CONCLUSION: This study delineated the spectrum of brain anomalies in MWS and provided new insights into the role of ZEB2 in neurodevelopment.Genet Med advance online publication 10 November 2016.

Exome sequencing in children of women with skewed X-inactivation identifies atypical cases and complex phenotypes.

BACKGROUND: More than 100 X-linked intellectual disability (X-LID) genes have been identified to be involved in 10-15% of intellectual disability (ID). METHOD: To identify novel possible candidates, we selected 18 families with a male proband affected by isolated or syndromic ID. Pedigree and/or clinical presentation suggested an X-LID disorder. After exclusion of known genetic diseases, we identified seven cases whose mother showed a skewed X-inactivation (>80%) that underwent whole exome sequencing (WES, 50X average depth). RESULTS: WES allowed to solve the genetic basis in four cases, two of which (Coffin-Lowry syndrome, RPS6K3 gene; ATRX syndrome, ATRX gene) had been missed by previous clinical/genetics tests. One further ATRX case showed a complex phenotype including pontocerebellar atrophy (PCA), possibly associated to an unidentified PCA gene mutation. In a case with suspected Lujan-Fryns syndrome, a c.649C>T (p.Pro217Ser) MECP2 missense change was identified, likely explaining the neurological impairment, but not the marfanoid features, which were possibly associated to the p.Thr1020Ala variant in fibrillin 1. Finally, a c.707T>G variant (p.Phe236Cys) in the DMD gene was identified in a patient retrospectively recognized to be affected by Becker muscular dystrophy (BMD, OMIM 300376). CONCLUSION: Overall, our data show that WES may give hints to solve complex ID phenotypes with a likely X-linked transmission, and that a significant proportion of these orphan conditions might result from concomitant mutations affecting different clinically associated genes.

Endocrinological Abnormalities Are a Main Feature of 17p13.1 Microduplication Syndrome: A New Case and Literature Review.

To date, 5 cases of 17p13.1 microduplications have been described in the literature. Intellectual disability was reported as the core feature, together with minor facial dysmorphisms and obesity, but a characteristic phenotype for 17p13.1 microduplication has not been delineated. Here, we describe a patient with a 1.56-Mb de novo duplication in 17p13.1, affected by mild intellectual disability, facial dysmorphisms, obesity, and diabetes. By comparing the different phenotypes of currently described cases, we delineated the main clinical features of 17p13.1 microduplication syndrome. All patients described to date had variable facial dysmorphisms; therefore, it was difficult to define a common facial gestalt. Furthermore, we stress endocrinological abnormalities as important features and the need to monitor these over time.

Whole exome sequencing is necessary to clarify ID/DD cases with de novo copy number variants of uncertain significance: Two proof-of-concept examples.

Whole exome sequencing (WES) is a powerful tool to identify clinically undefined forms of intellectual disability/developmental delay (ID/DD), especially in consanguineous families. Here we report the genetic definition of two sporadic cases, with syndromic ID/DD for whom array-Comparative Genomic Hybridization (aCGH) identified a de novo copy number variant (CNV) of uncertain significance. The phenotypes included microcephaly with brachycephaly and a distinctive facies in one proband, and hypotonia in the legs and mild ataxia in the other. WES allowed identification of a functionally relevant homozygous variant affecting a known disease gene for rare syndromic ID/DD in each proband, that is, c.1423C>T (p.Arg377*) in the Trafficking Protein Particle Complex 9 (TRAPPC9), and c.154T>C (p.Cys52Arg) in the Very Low Density Lipoprotein Receptor (VLDLR). Four mutations affecting TRAPPC9 have been previously reported, and the present finding further depicts this syndromic form of ID, which includes microcephaly with brachycephaly, corpus callosum hypoplasia, facial dysmorphism, and overweight. VLDLR-associated cerebellar hypoplasia (VLDLR-CH) is characterized by non-progressive congenital ataxia and moderate-to-profound intellectual disability. The c.154T>C (p.Cys52Arg) mutation was associated with a very mild form of ataxia, mild intellectual disability, and cerebellar hypoplasia without cortical gyri simplification. In conclusion, we report two novel cases with rare causes of autosomal recessive ID, which document how interpreting de novo array-CGH variants represents a challenge in consanguineous families; as such, clinical WES should be considered in diagnostic testing. © 2016 Wiley Periodicals, Inc.

A novel 3q29 deletion associated with autism, intellectual disability, psychiatric disorders, and obesity.

Copy number variation (CNV) has been associated with a variety of neuropsychiatric disorders, including intellectual disability/developmental delay (ID/DD), autism spectrum disorder (ASD), and schizophrenia (SCZ). Often, individuals carrying the same pathogenic CNV display high clinical variability. By array-CGH analysis, we identified a novel familial 3q29 deletion (1.36 Mb), centromeric to the 3q29 deletion region, which manifests with variable expressivity. The deletion was identified in a 3-year-old girl diagnosed with ID/DD and autism and segregated in six family members, all affected by severe psychiatric disorders including schizophrenia, major depression, anxiety disorder, and personality disorder. All individuals carrying the deletion were overweight or obese, and anomalies compatible with optic atrophy were observed in three out of four cases examined. Amongst the 10 genes encompassed by the deletion, the haploinsufficiency of Optic Atrophy 1 (OPA1), associated with autosomal dominant optic atrophy, is likely responsible for the ophthalmological anomalies. We hypothesize that the haploinsufficiency of ATPase type 13A4 (ATP13A4) and/or Hairy/Enhancer of Split Drosophila homolog 1 (HES1) contribute to the neuropsychiatric phenotype, while HES1 deletion might underlie the overweight/obesity. In conclusion, we propose a novel contiguous gene syndrome due to a proximal 3q29 deletion variably associated with autism, ID/DD, psychiatric traits and overweight/obesity.

Phenotype and genotype in 103 patients with tricho-rhino-phalangeal syndrome.

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities, and subdivided in TRPS I, caused by mutations in TRPS1, and TRPS II, caused by a contiguous gene deletion affecting (amongst others) TRPS1 and EXT1. We performed a collaborative international study to delineate phenotype, natural history, variability, and genotype-phenotype correlations in more detail. We gathered information on 103 cytogenetically or molecularly confirmed affected individuals. TRPS I was present in 85 individuals (22 missense mutations, 62 other mutations), TRPS II in 14, and in 5 it remained uncertain whether TRPS1 was partially or completely deleted. Main features defining the facial phenotype include fine and sparse hair, thick and broad eyebrows, especially the medial portion, a broad nasal ridge and tip, underdeveloped nasal alae, and a broad columella. The facial manifestations in patients with TRPS I and TRPS II do not show a significant difference. In the limbs the main findings are short hands and feet, hypermobility, and a tendency for isolated metacarpals and metatarsals to be shortened. Nails of fingers and toes are typically thin and dystrophic. The radiological hallmark are the cone-shaped epiphyses and in TRPS II multiple exostoses. Osteopenia is common in both, as is reduced linear growth, both prenatally and postnatally. Variability for all findings, also within a single family, can be marked. Morbidity mostly concerns joint problems, manifesting in increased or decreased mobility, pain and in a minority an increased fracture rate. The hips can be markedly affected at a (very) young age. Intellectual disability is uncommon in TRPS I and, if present, usually mild. In TRPS II intellectual disability is present in most but not all, and again typically mild to moderate in severity. Missense mutations are located exclusively in exon 6 and 7 of TRPS1. Other mutations are located anywhere in exons 4-7. Whole gene deletions are common but have variable breakpoints. Most of the phenotype in patients with TRPS II is explained by the deletion of TRPS1 and EXT1, but haploinsufficiency of RAD21 is also likely to contribute. Genotype-phenotype studies showed that mutations located in exon 6 may have somewhat more pronounced facial characteristics and more marked shortening of hands and feet compared to mutations located elsewhere in TRPS1, but numbers are too small to allow firm conclusions.

Large cryptic genomic rearrangements with apparently normal karyotypes detected by array-CGH.

BACKGROUND: Conventional karyotyping (550 bands resolution) is able to identify chromosomal aberrations >5-10 Mb, which represent a known cause of intellectual disability/developmental delay (ID/DD) and/or multiple congenital anomalies (MCA). Array-Comparative Genomic Hybridization (array-CGH) has increased the diagnostic yield of 15-20%. RESULTS: In a cohort of 700 ID/DD cases with or without MCA, including 15 prenatal diagnoses, we identified a subgroup of seven patients with a normal karyotype and a large complex rearrangement detected by array-CGH (at least 6, and up to 18 Mb). FISH analysis could be performed on six cases and showed that rearrangements were translocation derivatives, indistinguishable from a normal karyotype as they involved a similar band pattern and size. Five were inherited from a parent with a balanced translocation, whereas two were apparently de novo. Genes spanning the rearrangements could be associated with some phenotypic features in three cases (case 3: DOCK8; case 4: GATA3, AKR1C4; case 6: AS/PWS deletion, CHRNA7), and in two, likely disease genes were present (case 5: NR2F2, TP63, IGF1R; case 7: CDON). Three of our cases were prenatal diagnoses with an apparently normal karyotype. CONCLUSIONS: Large complex rearrangements of up to 18 Mb, involving chromosomal regions with similar size and band appearance may be overlooked by conventional karyotyping. Array-CGH allows a precise chromosomal diagnosis and recurrence risk definition, further confirming this analysis as a first tier approach to clarify molecular bases of ID/DD and/or MCA. In prenatal tests, array-CGH is confirmed as an important tool to avoid false negative results due to karyotype intrinsic limit of detection.

Defining the phenotype associated with microduplication reciprocal to Sotos syndrome microdeletion.

NSD1 point mutations, submicroscopic deletions and intragenic deletions are the major cause of Sotos syndrome, characterized by pre-postnatal generalized overgrowth with advanced bone age, learning disability, seizures, distinctive facial phenotype. Reverse clinical phenotype due to 5q35 microduplication encompassing NSD1 gene has been reported so far in 27 cases presenting with delayed bone age, microcephaly, failure to thrive and seizures in some cases, further supporting a gene dosage effect of NSD1 on growth regulation and neurological functions. Here we depict the clinical presentation of three new cases with 5q35 microduplication outlining a novel syndrome characterized by microcephaly, short stature, developmental delay and in some cases delayed bone maturation, without any typical facial or osseous anomalies.

Progressive extreme heterotopic calcification.

The formation of ectopic calcifications in soft tissues can occur either sporadically or as a genetically determined condition, and is seen only infrequently. We report on a girl in whom widespread, rapidly progressive ectopic calcifications were detected shortly after birth. Calcifications became present around all joints, tendons and ligaments, but did not involve internal organs and skin, and eventually caused almost complete immobility of the child at 2 years. There were no other health problems and cognitive development was normal. We compare the manifestations in the child to the characteristics of known entities causing ectopic calcifications and conclude the child differs to each. Laboratory evaluation failed to identify autoimmune phenomena as well as calcium metabolism or other biochemical abnormalities; molecular studies did not identify occurrence of mutations in disease genes known to be involved in ectopic calcifications. We conclude the manifestations in the child represent an unreported entity of hitherto unknown etiology.

790 Kb microduplication in chromosome band 17p13.1 associated with intellectual disability, afebrile seizures, dysmorphic features, diabetes, and hypothyroidism.

We report a patient with a moderate mental retardation, afebrile seizure, mild dysmorphic features and type 2 diabetes mellitus with mild obesity and metabolic syndrome. Array-CGH analysis revealed a de novo 790-830 kb duplication on chromosome 17p13.1, not reported so far. Among the approximately 50 genes involved in the rearrangement, neuroligin 2 (NLGN2) and ephrin B3 (EFNB3) are candidates for the mental retardation phenotype. NLGN2 may therefore be a novel candidate gene for mental retardation or autistic spectrum disorder, joining other members of the neurexin/neuroligin network. Moreover, GLUT4, a member of the solute carrier family 2, may play a role in the patient's type 2 diabetes.

A restricted spectrum of mutations in the SMAD4 tumor-suppressor gene underlies Myhre syndrome.

Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor ß and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth.

Characterization of a 8q21.11 microdeletion syndrome associated with intellectual disability and a recognizable phenotype.

We report eight unrelated individuals with intellectual disability and overlapping submicroscopic deletions of 8q21.11 (0.66-13.55 Mb in size). The deletion was familial in one and simplex in seven individuals. The phenotype was remarkably similar and consisted of a round face with full cheeks, a high forehead, ptosis, cornea opacities, an underdeveloped alae, a short philtrum, a cupid's bow of the upper lip, down-turned corners of the mouth, micrognathia, low-set and prominent ears, and mild finger and toe anomalies (camptodactyly, syndactyly, and broadening of the first rays). Intellectual disability, hypotonia, decreased balance, sensorineural hearing loss, and unusual behavior were frequently observed. A high-resolution oligonucleotide array showed different proximal and distal breakpoints in all of the individuals. Sequencing studies in three of the individuals revealed that proximal and distal breakpoints were located in unique sequences with no apparent homology. The smallest region of overlap was a 539.7 kb interval encompassing three genes: a Zinc Finger Homeobox 4 (ZFHX4), one microRNA of unknown function, and one nonfunctional pseudogen. ZFHX4 encodes a transcription factor expressed in the adult human brain, skeletal muscle, and liver. It has been suggested as a candidate gene for congenital bilateral isolated ptosis. Our results suggest that the 8q21.11 submicroscopic deletion represents a clinically recognizable entity and that a haploinsufficient gene or genes within the minimal deletion region could underlie this syndrome.

Mutation spectrum of MLL2 in a cohort of Kabuki syndrome patients.

BACKGROUND: Kabuki syndrome (Niikawa-Kuroki syndrome) is a rare, multiple congenital anomalies/mental retardation syndrome characterized by a peculiar face, short stature, skeletal, visceral and dermatoglyphic abnormalities, cardiac anomalies, and immunological defects. Recently mutations in the histone methyl transferase MLL2 gene have been identified as its underlying cause. METHODS: Genomic DNAs were extracted from 62 index patients clinically diagnosed as affected by Kabuki syndrome. Sanger sequencing was performed to analyze the whole coding region of the MLL2 gene including intron-exon junctions. The putative causal and possible functional effect of each nucleotide variant identified was estimated by in silico prediction tools. RESULTS: We identified 45 patients with MLL2 nucleotide variants. 38 out of the 42 variants were never described before. Consistently with previous reports, the majority are nonsense or frameshift mutations predicted to generate a truncated polypeptide. We also identified 3 indel, 7 missense and 3 splice site. CONCLUSIONS: This study emphasizes the relevance of mutational screening of the MLL2 gene among patients diagnosed with Kabuki syndrome. The identification of a large spectrum of MLL2 mutations possibly offers the opportunity to improve the actual knowledge on the clinical basis of this multiple congenital anomalies/mental retardation syndrome, design functional studies to understand the molecular mechanisms underlying this disease, establish genotype-phenotype correlations and improve clinical management.

Prenatal and postnatal growth retardation, microcephaly, developmental delay, and pigmentation abnormalities: Naegeli syndrome, dyskeratosis congenita, poikiloderma Clericuzio type, or separate entity?

Naegeli(-Franceschetti-Jadassohn) syndrome and Dermatopathia Pigmentosa Reticularis are allelic disorders, both characterized by a congenital generalized reticulate hyperpigmentation, palmoplantar hyperkeratosis and other ectodermal symptoms. The disorders differ in their primary pigmentation localization and hair and dental manifestations. They resemble Dyskeratosis Congenita and Poikiloderma Clericuzio type in many of the skin changes, but especially the presence of leukoplakia and bone marrow disfunctioning in the first, and of telangiectasias, generalized hyperkeratosis of palms and soles, and nail pachyonychia in the latter are distinguishing features. Here we present two unrelated patients who have prenatal and postnatal growth retardation, microcephaly, developmental delay, generalized reticulate hyperpigmentation, hypohidrosis, absent fingertip prints, and absent palmoplantar hyperkeratosis. The patients differ in nail manifestations and hair colour. No Keratin14 mutation or genomic imbalance at CGHarray could be found in either of them. Although their phenotype overlaps with Naegeli syndrome, dermatopathia pigmentosa reticularis, dyskeratosis congenita and poikiloderma Clericuzio type, the differences in ectodermal manifestations, immunological functioning, growth pattern and cognition may indicate the presence of a separate entity.

Familial occurrence of ptosis, nasal speech, prominent ears, hand anomalies and learning problems.

We describe a four-generation family in whom 5 members show the combination of a large head, ptosis, nasal speech that sometimes goes along with a cleft palate, full cheeks, small mouth, and prominent ears, and who also have learning problems. We evaluated three affected members in detail and found them to have in addition a partial cutaneous syndactyly between the third and fourth fingers, an increased distance between second and third finger, and a decreased smell. We have not been unable to find other patients described in literature with the same combination of features, and suggest this to represent a hitherto unrecognizable entity. Pattern of inheritance is likely to be autosomal dominant.

Subtelomeric FISH analysis in 76 patients with syndromic developmental delay/intellectual disability.

BACKGROUND: Intellectual disability affects approximately 1 to 3% of the general population. The etiology is still poorly understood and it is estimated that one-half of the cases are due to genetic factors. Cryptic subtelomeric aberrations have been found in roughly 5 to 7% of all cases. METHODS: We performed a subtelomeric FISH analysis on 76 unrelated children with normal standard karyotype ascertained by developmental delay or intellectual disability, associated with congenital malformations, and/or facial dysmorphisms. RESULTS: Ten cryptic chromosomal anomalies have been identified in the whole cohort (13,16%), 8 in the group of patients characterized by developmental delay or intellectual disability associated with congenital malformations and facial dysmorphisms, 2 in patients with developmental delay or intellectual disability and facial dysmorphisms only. CONCLUSION: We demonstrate that a careful clinical examination is a very useful tool for pre-selection of patients for genomic analysis, clearly enhancing the chromosomal anomaly detection rate. Clinical features of most of these patients are consistent with the corresponding emerging chromosome phenotypes, pointing out these new clinical syndromes associated with specific genomic imbalances.

Phenotype resembling Donnai-Barrow syndrome in a patient with 9qter;16qter unbalanced translocation.

We describe a 3-year-old boy with complete agenesis of corpus callosum, developmental delay/mental retardation, anterior diaphragmatic hernia, Morgagni type, severe hypermetropia, and facial dysmorphism suggesting the diagnosis of Donnai-Barrow syndrome. Subtelomeric FISH analysis revealed a paternally-derived t(9;16) (q34.3;q24.3) translocation with partial 9q monosomy and partial 16q trisomy. As some facial features resemble the 9q emerging phenotype, we suggest the hypothesis that some patients with Donnai-Barrow syndrome might be abscribed to 9q terminal deletion.