POU3F3-related disorder: Defining the phenotype and expanding the molecular spectrum.

POU3F3 variants cause developmental delay, behavioral problems, hypotonia and dysmorphic features. We investigated the phenotypic and genetic landscape, and genotype-phenotype correlations in individuals with POU3F3-related disorders. We recruited unpublished individuals with POU3F3 variants through international collaborations and obtained updated clinical data on previously published individuals. Trio exome sequencing or single exome sequencing followed by segregation analysis were performed in the novel cohort. Functional effects of missense variants were investigated with 3D protein modeling. We included 28 individuals (5 previously published) from 26 families carrying POU3F3 variants; 23 de novo and one inherited from an affected parent. Median age at study inclusion was 7.4 years. All had developmental delay mainly affecting speech, behavioral difficulties, psychiatric comorbidities and dysmorphisms. Additional features included gastrointestinal comorbidities, hearing loss, ophthalmological anomalies, epilepsy, sleep disturbances and joint hypermobility. Autism, hearing and eye comorbidities, dysmorphisms were more common in individuals with truncating variants, whereas epilepsy was only associated with missense variants. In silico structural modeling predicted that all (likely) pathogenic variants destabilize the DNA-binding region of POU3F3. Our study refined the phenotypic and genetic landscape of POU3F3-related disorders, it reports the functional properties of the identified pathogenic variants, and delineates some genotype-phenotype correlations.

Identifying phenotypic expansions for congenital diaphragmatic hernia plus (CDH+) using DECIPHER data.

Congenital diaphragmatic hernia (CDH) can occur in isolation or in conjunction with other birth defects (CDH+). A molecular etiology can only be identified in a subset of CDH cases. This is due, in part, to an incomplete understanding of the genes that contribute to diaphragm development. Here, we used clinical and molecular data from 36 individuals with CDH+ who are cataloged in the DECIPHER database to identify genes that may play a role in diaphragm development and to discover new phenotypic expansions. Among this group, we identified individuals who carried putatively deleterious sequence or copy number variants affecting CREBBP, SMARCA4, UBA2, and USP9X. The role of these genes in diaphragm development was supported by their expression in the developing mouse diaphragm, their similarity to known CDH genes using data from a previously published and validated machine learning algorithm, and/or the presence of CDH in other individuals with their associated genetic disorders. Our results demonstrate how data from DECIPHER, and other public databases, can be used to identify new phenotypic expansions and suggest that CREBBP, SMARCA4, UBA2, and USP9X play a role in diaphragm development.

Proximal variants in CCND2 associated with microcephaly, short stature, and developmental delay: A case series and review of inverse brain growth phenotypes.

Cyclin D2 (CCND2) is a critical cell cycle regulator and key member of the cyclin D2-CDK4 (DC) complex. De novo variants of CCND2 clustering in the distal part of the protein have been identified as pathogenic causes of brain overgrowth (megalencephaly, MEG) and severe cortical malformations in children including the megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndrome. Megalencephaly-associated CCND2 variants are localized to the terminal exon and result in accumulation of degradation-resistant protein. We identified five individuals from three unrelated families with novel variants in the proximal region of CCND2 associated with microcephaly, mildly simplified cortical gyral pattern, symmetric short stature, and mild developmental delay. Identified variants include de novo frameshift variants and a dominantly inherited stop-gain variant segregating with the phenotype. This is the first reported association between proximal CCND2 variants and microcephaly, to our knowledge. This series expands the phenotypic spectrum of CCND2-related disorders and suggests that distinct classes of CCND2 variants are associated with reciprocal effects on human brain growth (microcephaly and megalencephaly due to possible loss or gain of protein function, respectively), adding to the growing paradigm of inverse phenotypes due to dysregulation of key brain growth genes.

Microcephalic osteodysplastic primordial dwarfism type II and pachygyria: Morphometric analysis in a 2-year-old girl.

Microcephalic osteodysplastic primordial dwarfism (MOPD) type II is a rare disorder characterized by skeletal dysplasia, severe proportionate short stature, insulin resistance and cerebrovascular abnormalities including cerebral aneurysms and moyamoya disease. MOPD type II is caused by mutations in the pericentrin (PCNT) gene, which encodes a protein involved in centrosomes function. We report a 2 year old girl affected by MOPD type II caused by two compound heterozygous loss-of-function variants in PCNT gene, of which one is a novel variant (c.5304delT; p.Gly1769AlafsTer34). The patient presented atypical brain magnetic resonance imaging (MRI) findings consistent with pachygyria. This was confirmed by morphometric analysis of cortical thickness (CT) and gyrification index by comparing MRI data of the patient with a group of eight age-matched healthy controls. The statistical analysis revealed a significant and diffuse increase of CT with an anterior-predominant pattern and diffuse reduced gyrification (p < .05). These findings provide new evidences to the emergent concept that malformations of cortical development are complex disorders and that new genetic findings contribute to the fading of classification borders.

TUBB3 E410K syndrome: Case report and review of the clinical spectrum of TUBB3 mutations.

The tubulinopathies refer to a wide range of brain malformations caused by mutations in one of the seven genes encoding different tubulin's isotypes. The ß-tubulin isotype III (TUBB3) gene has a primary function in nervous system development and axon generation and maintenance, due to its neuron-specific expression pattern. A recurrent heterozygous mutation, c.1228G > A; p.E410K, in TUBB3 gene is responsible of a rare disorder clinically characterized by congenital fibrosis of the extraocular muscle type 3 (CFEOM3), intellectual disability and a wide range of neurological and endocrine abnormalities. Other mutations have been described spanning the entire gene and genotype-phenotype correlations have been proposed. We report on a 3-year-old boy in whom clinical exome sequencing allowed to identify a de novo TUBB3 E410K mutation as the molecular cause underlying a complex phenotype characterized by a severe bilateral palpebral ptosis refractory to eye surgery, psychomotor delay, absent speech, hypogonadism, celiac disease, and cyclic vomiting. Brain MRI revealed thinning of the corpus callosum with no evidence of malformation cortical dysplasia. We reviewed available records of patients with TUBB3 E410K mutation and compared their phenotype with the clinical outcome of patients with other mutations in TUBB3 gene. The present study confirms that TUBB3 E410K results in a clinically recognizable phenotype, unassociated to the distinct cortical dysplasia caused by other mutations in the same gene. Early molecular characterization of TUBB3 E410K syndrome is critical for targeted genetic counseling and prompt prospective care in term of neurological, ophthalmological, endocrine, and gastrointestinal follow-up.

POGZ-related epilepsy: Case report and review of the literature.

POGZ (# 614787) encodes a multidomain nuclear protein involved in transcriptional regulation and its defective function has been recently associated with a syndromic neurodevelopmental disorder, known as White-Sutton syndrome (# 616364). While originally epileptic seizures were unreported, it seems that epilepsy represents a recurrent feature in affected subjects. Few data, however, are available on electroclinical features of POGZ-related epilepsy. We report a 5-year-old girl with a de novo inactivating POGZ mutation with a complex neurological phenotype characterized by hypotonia, severe developmental delay, and paroxysmal epileptic and nonepileptic events. Comparing this patient with the previously reported nine cases exhibiting epilepsy as associated feature, we detected that epilepsy onset is mostly during infancy (1-4 years of age), with both focal and generalized seizures. EEGs reveal that epileptic abnormalities mainly are localized in the frontal regions, and seizure control might be reached with one or multiple antiepileptic drugs. Besides dysmorphic features and other comorbidities (microcephaly, intellectual disability, absent speech, sensorineural hearing loss, and autistic spectrum disorder) major brain MR features include cortical and cerebellar atrophy, delayed myelination, and brainstem hypoplasia. Although the small number of patients reported, we were able to delineate primary electroclinical epileptic phenotype related to POGZ mutations. This would be crucial for an early identification and management of the condition.

Expanding the clinical spectrum associated with PACS2 mutations.

Whole exome sequencing (WES) has led to the understanding of the molecular events affecting neurodevelopment in an extremely diverse clinical context, including diseases with intellectual disability (ID) associated with variable central nervous system (CNS) malformations, and developmental and epileptic encephalopathies (DEEs). Recently, PACS2 mutations have been causally linked to a DEE with cerebellar dysgenesis and facial dysmorphism. All known patients presented with a recurrent de novo missense mutation, c.625G>A (p.Glu209Lys). Here, we report on a 7-year-old boy with DEE, cerebellar dysgenesis, facial dysmorphism and postnatal growth delay, apparently not fitting with any recognized disorder. WES disclosed a de novo novel missense PACS2 variant, c.631G>A (p.Glu211Lys), as the molecular cause of this complex phenotype. We provide a detailed clinical characterization of this patient, and analyse the available clinical data of individuals with PACS2 mutations to delineate more accurately the clinical spectrum associated with this recently described syndrome. Our study expands the clinical and molecular spectrum of PACS2 mutations. Overview of the available clinical data allow to delineate the condition associated with PACS2 mutations as a variable trait, in which the key features are represented by moderate to severe ID, cerebellar dysgenesis and other CNS malformations, reduced growth, and facial dysmorphism.

First Report of Low-Rate Mosaicism for 20q11.21q12 Deletion and Delineation of the Associated Disorder.

Interstitial deletions of the long arm of chromosome 20 are very rare, with only 12 reported patients harboring the 20q11.2 microdeletion and presenting a disorder characterized by psychomotor and growth delay, dysmorphisms, and brachy-/clinodactyly. We describe the first case of mosaic 20q11.2 deletion in a 5-year-old girl affected by mild psychomotor delay, feeding difficulties, growth retardation, craniofacial dysmorphisms, and finger anomalies. SNP array analysis disclosed 20% of cells with a 20q11.21q12 deletion, encompassing the 20q11.2 minimal critical region and the 3 OMIM disease-causing genes GDF5, EPB41L1, and SAMHD1. We propose a pathogenic role of other genes mapping outside the small region of overlap, in particular GHRH (growth hormone releasing hormone), whose haploinsufficiency could be responsible for the prenatal onset of growth retardation which is shared by half of these patients. Our patient highlights the utility of chromosomal microarray analysis to identify low-level mosaicism.

Unclassifiable pattern of hypopigmentation in a patient with mosaic partial 12p tetrasomy without Pallister-Killian syndrome.

Pallister-Killian syndrome (PKS-#OMIM601803) is a multisystem developmental disorder typically due to the presence of an aneuploidy cell line, consisting of a supernumerary tetrasomic chromosomal marker (SCM) arisen from the short arm of chromosome 12 (12p isochromosome). The clinical phenotype, which is strictly related to the percentage and tissue distribution of aneuploid cells, is characterized by craniofacial dysmorphisms, pigmentary skin anomalies, limb shortening, congenital heart defects, diaphragmatic hernia, hypotonia, intellectual disability, and epilepsy. We report on a 4 year-old girl harboring a 12p partial isochromosome, involving the PKS critical region, affecting about 70% of circulating lymphocytes, urine, and saliva cells and fibroblast from a hyperpigmented skin spot, and 100% of fibroblasts from a hypopigmented skin spot. Interestingly, despite the high proportion of affected cells this patient did not present with PKS, and a pattern of linear and patchy pigmentary mosaicism was the sole clinical manifestation. The present observation suggests that partial 12p SCM can also result in mild phenotypes, and its prevalence in the human population could have been underestimated. Accurate dermatologic evaluation could be a major handle for genetic testing.

Interstitial 10q21.1q23.31 Duplication due to Meiotic Recombination of a Paternal Balanced Complex Rearrangement: Cytogenetic and Molecular Characterization.

Complex chromosomal rearrangements (CCRs) are structural aberrations involving more than 2 chromosomal breakpoints. They are associated with different outcomes depending on the deletion/duplication of genomic material, gene disruption, or position effects. Balanced CCRs can also undergo missegregation during meiotic division, leading to unbalanced derivative chromosomes and, in some cases, to affected offspring. We report on a patient presenting with developmental and speech delay, growth retardation, microcephaly, hypospadias, and dysmorphic features, harboring an interstitial 10q21.1q23.31 duplication, due to recombination of a paternal CCR. Application of several cytogenetic and molecular techniques allowed determining the biological bases of the rearrangement, understanding the underlying chromosomal mechanism, and assessing the reproductive risk.

New patients with Temple syndrome caused by 14q32 deletion: Genotype-phenotype correlations and risk of thyroid cancer.

Temple syndrome (TS) is caused by abnormal expression of genes at the imprinted locus 14q32. A subset of TS patients carry 14q32 deletions of paternal origin. We aimed to define possible genotype-phenotype correlations and to highlight the prevalence of thyroid dysfunction, which is a previously unreported feature of TS. We described four new patients who carry deletions of paternal origin at 14q32 detected by array-CGH and reviewed nine patients reported in the medical literature. We compared clinical features with respect to deletion size and position. Expression of DLK1 is altered in all the patients with TS, but intellectual disability (ID) is present only in patients with larger deletions extending proximally to the imprinted locus. This study led to the identification of an ID "critical region" containing four annotated genes including YY1 as the strongest candidate. Furthermore, we described three patients with thyroid dysfunction, which progressed to papillary carcinoma at a very young age in two of them. We conclude that DLK1 loss of function is likely to be responsible for the core features of TS, while haploinsufficiency of a gene outside the imprinted region causes ID. Thyroid cancer may be an unrecognized feature and monitoring for thyroid dysfunction should thus be considered in TS patients.

Association of DiGeorge anomaly and caudal dysplasia sequence in a neonate born to a diabetic mother.

AIM/OBJECTIVE: Several studies have demonstrated a significantly increased risk of specific patterns of congenital anomalies in infants born to diabetic mothers. In particular, caudal dysplasia sequence has been linked to pregnancy complicated by maternal diabetes. In addition, several cases of infants born to diabetic mothers presenting with features of DiGeorge anomaly have been reported. Infants with DiGeorge anomaly can display additional manifestations within the spectrum of caudal dysplasia sequence, including vertebral anomalies and renal agenesis. METHODS: We report a neonate presenting with the co-occurrence of features of both DiGeorge anomaly and caudal dysplasia sequence, born to a mother with poorly controlled insulin-dependent diabetes. RESULTS: The patient was affected by truncus arteriosus type A1 and hypertrophic cardiomyopathy. CONCLUSION: Maternal diabetes can cause a spectrum of manifestations, expressing with isolated DiGeorge anomaly or caudal dysplasia sequence, with intermediate phenotypes or with the co-occurrence of both the congenital anomalies in the same patient. The present observations argue for a feasible link between truncus arteriosus with hypertrophic cardiomyopathy, DiGeorge anomaly, and maternal diabetes.

SOS1 mutations in Noonan syndrome: molecular spectrum, structural insights on pathogenic effects, and genotype-phenotype correlations.

Noonan syndrome (NS) is among the most common nonchromosomal disorders affecting development and growth. NS is caused by aberrant RAS-MAPK signaling and is genetically heterogeneous, which explains, in part, the marked clinical variability documented for this Mendelian trait. Recently, we and others identified SOS1 as a major gene underlying NS. Here, we explored further the spectrum of SOS1 mutations and their associated phenotypic features. Mutation scanning of the entire SOS1 coding sequence allowed the identification of 33 different variants deemed to be of pathological significance, including 16 novel missense changes and in-frame indels. Various mutation clusters destabilizing or altering orientation of regions of the protein predicted to contribute structurally to the maintenance of autoinhibition were identified. Two previously unappreciated clusters predicted to enhance SOS1's recruitment to the plasma membrane, thus promoting a spatial reorientation of domains contributing to inhibition, were also recognized. Genotype-phenotype analysis confirmed our previous observations, establishing a high frequency of ectodermal anomalies and a low prevalence of cognitive impairment and reduced growth. Finally, mutation analysis performed on cohorts of individuals with nonsyndromic pulmonic stenosis, atrial septal defects, and ventricular septal defects excluded a major contribution of germline SOS1 lesions to the isolated occurrence of these cardiac anomalies.

A restricted spectrum of NRAS mutations causes Noonan syndrome.

Noonan syndrome, a developmental disorder characterized by congenital heart defects, reduced growth, facial dysmorphism and variable cognitive deficits, is caused by constitutional dysregulation of the RAS-MAPK signaling pathway. Here we report that germline NRAS mutations conferring enhanced stimulus-dependent MAPK activation account for some cases of this disorder. These findings provide evidence for an obligate dependency on proper NRAS function in human development and growth.

Germline BRAF mutations in Noonan, LEOPARD, and cardiofaciocutaneous syndromes: molecular diversity and associated phenotypic spectrum.

Noonan, LEOPARD, and cardiofaciocutaneous syndromes (NS, LS, and CFCS) are developmental disorders with overlapping features including distinctive facial dysmorphia, reduced growth, cardiac defects, skeletal and ectodermal anomalies, and variable cognitive deficits. Dysregulated RAS-mitogen-activated protein kinase (MAPK) signal traffic has been established to represent the molecular pathogenic cause underlying these conditions. To investigate the phenotypic spectrum and molecular diversity of germline mutations affecting BRAF, which encodes a serine/threonine kinase functioning as a RAS effector frequently mutated in CFCS, subjects with a diagnosis of NS (N=270), LS (N=6), and CFCS (N=33), and no mutation in PTPN11, SOS1, KRAS, RAF1, MEK1, or MEK2, were screened for the entire coding sequence of the gene. Besides the expected high prevalence of mutations observed among CFCS patients (52%), a de novo heterozygous missense change was identified in one subject with LS (17%) and five individuals with NS (1.9%). Mutations mapped to multiple protein domains and largely did not overlap with cancer-associated defects. NS-causing mutations had not been documented in CFCS, suggesting that the phenotypes arising from germline BRAF defects might be allele specific. Selected mutant BRAF proteins promoted variable gain of function of the kinase, but appeared less activating compared to the recurrent cancer-associated p.Val600Glu mutant. Our findings provide evidence for a wide phenotypic diversity associated with mutations affecting BRAF, and occurrence of a clinical continuum associated with these molecular lesions.