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Dieuwerke R Dijk,1 Gianni Bocca,2 Conny M van Ravenswaaij-Arts11Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 2Department of Pediatrics, Beatrix Children’s Hospital, University of Groningen, University Medical Center Groningen, Groningen, The NetherlandsAbstract: CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital hypoplasia, Ear anomalies including hearing loss) syndrome is a rare syndrome with an incidence of approximately 1:15,000 newborns. It is caused by pathogenic variants in the CHD7 gene and clinically characterized by a wide range of anomalies with variable expression. Growth retardation affects 60–72% of children with CHARGE syndrome, making it one of the most prominent medical issues in the syndrome. Growth retardation in CHARGE syndrome is thought to be multifactorial and can be influenced by almost all co-morbidities, requiring a multidisciplinary approach to the different medical problems. In this systematic review, we describe what is currently known about growth in CHARGE syndrome and how it is influenced by commonly seen clinical problems including feeding difficulties, hypogonadotropic hypogonadism and growth hormone deficiency. Furthermore, we provide recommendations for a multidisciplinary approach.Keywords: CHARGE syndrome, growth, short stature, multidisciplinary, hypogonadotropic hypogonadism

BACKGROUND The prevalence of chromosomal abnormalities is assumed to be higher in infertile men and inversely correlated with sperm concentration. Although guidelines advise karyotyping infertile men, karyotyping is costly, therefore it would be of benefit to identify men with the highest risk of chromosomal abnormalities, possibly by using parameters other than sperm concentration. The aim of this study was to evaluate several clinical parameters in azoospermic and non-azoospermic men, in order to assess the prevalence of chromosomal abnormalities in different subgroups of infertile men. METHODS In a retrospective cohort of 1223 azoospermic men and men eligible for ICSI treatment, we studied sperm parameters, hormone levels and medical history for an association with chromosomal abnormalities. RESULTS The prevalence of chromosomal abnormalities in the cohort was 3.1%. No association was found between chromosomal abnormalities and sperm volume, concentration, progressive motility or total motile sperm count. Azoospermia was significantly associated with the presence of a chromosomal abnormality [15.2%, odds ratio (OR) 7.70, P < 0.001]. High gonadotrophin levels were also associated with an increased prevalence of chromosomal abnormalities (OR 2.96, P = 0.013). Azoospermic men with a positive andrologic history had a lower prevalence of chromosomal abnormalities than azoospermic men with an uneventful history (OR 0.28, P = 0.047). In non-azoospermic men, we found that none of the studied variables were associated with the prevalence of chromosomal abnormalities. CONCLUSIONS We show that the highest prevalence of chromosomal abnormalities is found in hypergonadotrophic azoospermic men with an uneventful andrologic history. [ABSTRACT FROM AUTHOR]

Purpose: Postsynaptic density protein-95 (PSD-95), encoded by DLG4, regulates excitatory synaptic function in the brain. Here we present the clinical and genetic features of 53 patients (42 previously unpublished) with DLG4 variants., Methods: The clinical and genetic information were collected through GeneMatcher collaboration. All the individuals were investigated by local clinicians and the gene variants were identified by clinical exome/genome sequencing., Results: The clinical picture was predominated by early onset global developmental delay, intellectual disability, autism spectrum disorder, and attention deficit-hyperactivity disorder, all of which point to a brain disorder. Marfanoid habitus, which was previously suggested to be a characteristic feature of DLG4-related phenotypes, was found in only nine individuals and despite some overlapping features, a distinct facial dysmorphism could not be established. Of the 45 different DLG4 variants, 39 were predicted to lead to loss of protein function and the majority occurred de novo (four with unknown origin). The six missense variants identified were suggested to lead to structural or functional changes by protein modeling studies., Conclusion: The present study shows that clinical manifestations associated with DLG4 overlap with those found in other neurodevelopmental disorders of synaptic dysfunction; thus, we designate this group of disorders as DLG4-related synaptopathy.

Aneuploidy mosaicism involving two complementary different autosomal trisomy cell lines is extremely rare. Although a mosaic double trisomy 8/trisomy 21 has been described in literature, this is the first report of Warkany (+8)-Down (+21) syndrome due to two complementary mosaic trisomy cell lines. The phenotype of the male patient with Warkany-Down syndrome includes upslanting palpebral fissures, hypertelorism, small low-set ears with unilateral aural stenosis, large and broad hands and feet with deep palmar and plantar creases, bilateral cryptorchidism, generalized mild hypotonia and transient neonatal thrombocytopenia. At the age of two years, his developmental quotient is around 50. His height, weight and head circumference are below the third centile. We speculate on the mechanism of origin of the complementary trisomy cell lines based on molecular cytogenetic studies that showed no evidence for a chimera., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: Rapid diagnostic whole-genome sequencing has been explored in critically ill newborns, hoping to improve their clinical care and replace time-consuming and/or invasive diagnostic testing. A previous retrospective study in a research setting showed promising results with diagnoses in 57%, but patients were highly selected for known and likely Mendelian disorders. The aim of our prospective study was to assess the speed and yield of rapid targeted genomic diagnostics for clinical application., Methods: We included 23 critically ill children younger than 12 months in ICUs over a period of 2 years. A quick diagnosis could not be made after routine clinical evaluation and diagnostics. Targeted analysis of 3426 known disease genes was performed by using whole-genome sequencing data. We measured diagnostic yield, turnaround times, and clinical consequences., Results: A genetic diagnosis was obtained in 7 patients (30%), with a median turnaround time of 12 days (ranging from 5 to 23 days). We identified compound heterozygous mutations in the EPG5 gene (Vici syndrome), the RMND1 gene (combined oxidative phosphorylation deficiency-11), and the EIF2B5 gene (vanishing white matter), and homozygous mutations in the KLHL41 gene (nemaline myopathy), the GFER gene (progressive mitochondrial myopathy), and the GLB1 gene (GM1-gangliosidosis). In addition, a 1p36.33p36.32 microdeletion was detected in a child with cardiomyopathy., Conclusions: Rapid targeted genomics combined with copy number variant detection adds important value in the neonatal and pediatric intensive care setting. It led to a fast diagnosis in 30% of critically ill children for whom the routine clinical workup was unsuccessful., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2017 by the American Academy of Pediatrics.)

This paper compares the power of the parallel group design, the matched-pairs design, and several options for the stepped wedge and delayed start designs for testing a possible effect of intranasal insulin with respect to placebo on developmental growth of children with a rare disorder like Phelan-McDermid syndrome. A subject-specific linear mixed effects model for the primary outcome developmental age in a longitudinal setting with five time points was assumed. Monte Carlo simulation studies with small sample sizes were applied since the rare disorder prohibits large trials. The stepped wedge designs, which were initially preferred for ethical reasons, appear to be competitive in power to other designs and were in some settings even the best. The assumed statistical model also demonstrates that all of the designs can be viewed as a stepped wedge or delayed treatment design. Our results show that the stepped wedge design is an appropriate alternative for randomized controlled trials on developmental growth with small numbers of participants under the formulated statistical conditions.

Purpose: Truncating mutations in the maternally imprinted, paternally expressed gene MAGEL2, which is located in the Prader-Willi critical region 15q11-13, have recently been reported to cause Schaaf-Yang syndrome, a Prader-Willi-like disease that manifests as developmental delay/intellectual disability, hypotonia, feeding difficulties, and autism spectrum disorder. The causality of the reported variants in the context of the patients' phenotypes was questioned, as MAGEL2 whole-gene deletions seem to cause little or no clinical phenotype., Methods: Here we report a total of 18 newly identified individuals with Schaaf-Yang syndrome from 14 families, including 1 family with 3 individuals found to be affected with a truncating variant of MAGEL2, 11 individuals who are clinically affected but were not tested molecularly, and a presymptomatic fetal sibling carrying the pathogenic MAGEL2 variant., Results: All cases harbor truncating mutations of MAGEL2, and nucleotides c.1990-1996 arise as a mutational hotspot, with 10 individuals and 1 fetus harboring a c.1996dupC (p.Q666fs) mutation and 2 fetuses harboring a c.1996delC (p.Q666fs) mutation. The phenotypic spectrum of Schaaf-Yang syndrome ranges from fetal akinesia to neurobehavioral disease and contractures of the small finger joints., Conclusion: This study provides strong evidence for the pathogenicity of truncating mutations of the paternal allele of MAGEL2, refines the associated clinical phenotypes, and highlights implications for genetic counseling for affected families.Genet Med 19 1, 45-52.

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology., Competing Interests: The authors have none to declare.

Phelan-McDermid syndrome (PMS) or 22q13.3 deletion syndrome is a rare neurodevelopmental disorder with at least 60 children and 35 adults diagnosed in the Netherlands. Clinical features are moderate to severe intellectual disability and behavioural problems in the autism spectrum. Other researchers had observed a beneficial effect of intranasal insulin on development and behaviour in a pilot study in six children with PMS. To validate this effect, we conducted a randomized, double-blind, placebo-controlled clinical trial using a stepped-wedge design. From March 2013 to June 2015, 25 children aged 1-16 years with a molecularly confirmed 22q13.3 deletion including the SHANK3 gene participated in the clinical trial for a period of 18 months. Starting 6 months before the trial, children were systematically assessed for cognitive, language and motor development and for adaptive, social and emotional behaviour every 6 months. The second, third and fourth assessments were followed by daily nose sprays containing either intranasal insulin or intranasal placebo for a 6-month period. A fifth assessment was done directly after the end of the trial. Intranasal insulin did not cause serious adverse events. It increased the level of developmental functioning by 0.4-1.4 months per 6-month period, but the effect was not statistically significant in this small group. We found a stronger effect of intranasal insulin, being significant for cognition and social skills, for children older than 3 years, who usually show a decrease of developmental growth. However, clinical trials in larger study populations are required to prove the therapeutic effect of intranasal insulin in PMS.

Background: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously., Results: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management., Conclusions: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

Mutations in CREBBP cause Rubinstein-Taybi syndrome. By using exome sequencing, and by using Sanger in one patient, CREBBP mutations were detected in 11 patients who did not, or only in a very limited manner, resemble Rubinstein-Taybi syndrome. The combined facial signs typical for Rubinstein-Taybi syndrome were absent, none had broad thumbs, and three had only somewhat broad halluces. All had apparent developmental delay (being the reason for molecular analysis); five had short stature and seven had microcephaly. The facial characteristics were variable; main characteristics were short palpebral fissures, telecanthi, depressed nasal ridge, short nose, anteverted nares, short columella, and long philtrum. Six patients had autistic behavior, and two had self-injurious behavior. Other symptoms were recurrent upper airway infections (n = 5), feeding problems (n = 7) and impaired hearing (n = 7). Major malformations occurred infrequently. All patients had a de novo missense mutation in the last part of exon 30 or beginning of exon 31 of CREBBP, between base pairs 5,128 and 5,614 (codons 1,710 and 1,872). No missense or truncating mutations in this region have been described to be associated with the classical Rubinstein-Taybi syndrome phenotype. No functional studies have (yet) been performed, but we hypothesize that the mutations disturb protein-protein interactions by altering zinc finger function. We conclude that patients with missense mutations in this specific CREBBP region show a phenotype that differs substantially from that in patients with Rubinstein-Taybi syndrome, and may prove to constitute one (or more) separate entities. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Objective: To evaluate whether central adrenal insufficiency (CAI) is present in CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital hypoplasia, and Ear abnormalities, including deafness) syndrome, a complex malformation disorder that includes central endocrine dysfunction., Study Design: Two cross-sectional studies were performed in Dutch (September 2013-February 2015) and Australian (January 2012-January 2014) CHARGE syndrome clinics. Twenty-seven Dutch and 19 Australian patients (aged 16 months-18 years) with genetically confirmed CHARGE syndrome were included. The low-dose adrenocorticotropin (ACTH) test was used to assess CAI in the Dutch cohort. A peak cortisol response less than 18.1 μg/dL (500 nmol/L) was suspected for CAI, and a glucagon stimulation test was performed for confirmation. Australian patients were screened by single measurements of ACTH and cortisol levels. If adrenal dysfunction was suspected, a standard-dose ACTH test was performed., Results: The low-dose ACTH test was performed in 23 patients (median age 8.4 [1.9-16.9] years). Seven patients showed an insufficient maximum cortisol level (10.3-17.6 μg/dL, 285-485 nmol/L), but CAI was confirmed by glucagon stimulation test in only 1 patient (maximum cortisol level 15.0 μg/dL, 415 nmol/L). In the Australian cohort, 15 patients (median age 9.1 [1.3-17.8] years) were screened, and none had CAI., Conclusions: CAI was not common in our cohorts, and routine testing of adrenal function in children with CHARGE syndrome is not indicated., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Numerous genes are associated with neurodevelopmental disorders such as intellectual disability and autism spectrum disorder (ASD), but their dysfunction is often poorly characterized. Here we identified dominant mutations in the gene encoding the transcriptional repressor and MeCP2 interactor switch-insensitive 3 family member A (SIN3A; chromosome 15q24.2) in individuals who, in addition to mild intellectual disability and ASD, share striking features, including facial dysmorphisms, microcephaly and short stature. This phenotype is highly related to that of individuals with atypical 15q24 microdeletions, linking SIN3A to this microdeletion syndrome. Brain magnetic resonance imaging showed subtle abnormalities, including corpus callosum hypoplasia and ventriculomegaly. Intriguingly, in vivo functional knockdown of Sin3a led to reduced cortical neurogenesis, altered neuronal identity and aberrant corticocortical projections in the developing mouse brain. Together, our data establish that haploinsufficiency of SIN3A is associated with mild syndromic intellectual disability and that SIN3A can be considered to be a key transcriptional regulator of cortical brain development.

CHD7 variants are a well-established cause of CHARGE syndrome, a disabling multi-system malformation disorder that is often associated with deafness, visual impairment and intellectual disability. Less severe forms of CHD7-related disease are known to exist, but the full spectrum of phenotypes remains uncertain. We identified a de novo missense variant in CHD7 in a family presenting with musculoskeletal abnormalities as the main manifestation of CHD7-related disease, representing a new phenotype. The proband presented with prominent scapulae, mild shoulder girdle weakness and only subtle dysmorphic features. Investigation revealed hypoplasia of the trapezius and sternocleidomastoid muscles and semicircular canal defects, but he did not fulfill diagnostic criteria for CHARGE syndrome. Although the shoulders are often sloping and anteverted in CHARGE syndrome, the underlying neuromuscular cause has never been investigated. This report expands the phenotypes associated with CHD7 mutations to include a musculoskeletal presentation, with hypoplasia of the shoulder and neck muscles. CHD7 should be considered in patients presenting in childhood with stable scapular winging, particularly if accompanied by dysmorphic features and balance difficulties.

CHARGE syndrome is a dominant disorder characterized by ocular colobomata, heart defects, choanal atresia, retardation of growth and development, genital hypoplasia, and ear abnormalities including deafness and vestibular disorders. The majority of individuals with CHARGE have pathogenic variants in the gene encoding CHD7, a chromatin remodeling protein. Here, we present a 15-year-old girl with clinical features of CHARGE syndrome and a de novo 6.5 Mb gain of genomic material at 2p25.3-p25.2. The duplicated region contained 24 genes, including the early and broadly expressed transcription factor gene SOX11. Analysis of 28 other patients with CHARGE showed no SOX11 copy number changes or pathogenic sequence variants. To our knowledge, this child's chromosomal abnormality is unique and represents the first co-occurrence of duplication 2p25 and clinical features of CHARGE syndrome. We compare our patient's phenotype to ten previously published patients with isolated terminal duplication 2p, and elaborate on the clinical diagnosis of CHARGE in the context of atypical genetic findings., Competing Interests: The authors declare no conflicts of interest., (© 2016 Wiley Periodicals, Inc.)

We describe an 18-year-old male patient with myoclonic astatic epilepsy (MAE), moderate to severe intellectual disability, behavioural problems, several dysmorphisms and a 1.2-Mb de novo deletion on chromosome 16p11.2. This deletion results in haploinsufficiency of STX1B and other genes. Recently, variants in the STX1B gene have been associated with a wide spectrum of fever-related epilepsies ranging from single febrile seizures to severe epileptic encephalopathies. Two previously reported patients with a STX1B missense variant or deletion were diagnosed with MAE. Our observation of a STX1B deletion in a third patient with MAE therefore supports that STX1B gene variants or deletions can be involved in the aetiology of MAE. Furthermore, STX1B encodes for syntaxin-1B, of which interaction with the protein encoded by the STXBP1 gene is essential for the regulation of the synaptic transmission of neurotransmitters. STXBP1 gene variants have been identified in patients with many different types of epilepsy, including Dravet syndrome and epileptic encephalopathies, suggesting STX1B plays a similar role. We recommend that analysis of STX1B should be considered in the diagnostic work-up of individuals with MAE., (Copyright © 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Background: Phelan-McDermid syndrome (PMS) or 22q13.3 deletion syndrome is characterized by global developmental delay, cognitive deficits, and behaviour in the autism spectrum. Knowledge about developmental and behavioural characteristics of this rare chromosomal disorder is still limited despite a rapid growing number of diagnoses. Our aim was to study a new and relatively large cohort to further characterize the developmental phenotype of children with PMS., Methods: We performed a descriptive study of children with a 22q13.3 deletion including SHANK3, aged 8 to 178 months, who were systematically (n = 34) and longitudinally (n = 29) assessed with standardized instruments: Bayley Scales of Infant and Toddler Development, third edition; Wechsler Preschool and Primary Scale of Intelligence, third edition; and Vineland Screener for Social and Adaptive Behavior., Results: Maximal developmental functioning ranged from 34 to 52 months depending on the developmental domain. In general, children performed poorest in the domain of language and best on the domain of motor (young children) or cognitive development (older children). At the individual level, 25 % scored better for receptive and 18 % for expressive language, whereas 22 % scored better for fine and 33 % for gross motor function. Developmental quotients were higher in younger children and decreased with age for all developmental domains, with 38 % of the children showing no improvement of cognitive developmental functioning. Almost all children (33/34) had significant deficits in adaptive behaviour. Children with very small deletions, covering only the SHANK3, ACR, and RABL2B genes, had a more favourable developmental phenotype., Conclusions: Cognitive, motor, and especially language development were significantly impaired in all children with PMS but also highly variable and unpredictable. In addition, deficits in adaptive behaviour further hampered their cognitive development. Therefore, cognitive and behavioural characteristics should be evaluated and followed in each child with PMS to adapt supportive and therapeutic strategies to individual needs. Further research evaluating the relationship between deletion characteristics and the developmental phenotype is warranted to improve counselling of parents.

Phelan-McDermid syndrome (PMS) or 22q13.3 deletion syndrome is characterized by a variable degree of intellectual disability, impaired speech and language as well as social communicative skills and mild dysmorphic features. The SHANK3 gene is thought to be a major contributor to the phenotype. Apart from the syndrome-associated autistic features, symptoms from the bipolar spectrum can be discerned, in particular behavior instability and fluctuating mood culminating in a (hypo)manic state. In case of coincident major somatic events, a deteriorating course may occur. This study comprises seven adult patients (four females and three males; aged 21-44 years) with genetically proven PMS. Data from medical records were collected and extensive assessment of neuropsychological variables was performed to identify cognitive characteristics and their relation with psychopathology and treatment. All patients showed profound communication deficits and their developmental functioning ranged from 1.0 to 6.3 years. In addition, they had slow speed of information processing, impairment of attentional and executive functions and cognitive alexithymia. As to psychopathology, features from the affective and anxiety domains were prominent findings in these seven patients suggesting the presence of a bipolar spectrum disorder that could be effectively moderated with mood-stabilizing agents. Results are discussed in terms of the putative involvement of structural brain abnormalities, in particular cerebellar vermis hypoplasia and corpus callosum thinning and their cognitive and emotional sequelae. It is concluded that the treatment of 22q13.3-associated psychopathology should include prescription of mood-stabilizing agents in combination with individually tailored contextual neuropsychological measures., (© 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Background: Clinical and genetic heterogeneity in monogenetic disorders represents a major diagnostic challenge. Although the presence of particular clinical features may aid in identifying a specific cause in some cases, the majority of patients remain undiagnosed. Here, we investigated the utility of whole-exome sequencing as a diagnostic approach for establishing a molecular diagnosis in a highly heterogeneous group of patients with varied intellectual disability and microcephaly., Methods: Whole-exome sequencing was performed in 38 patients, including three sib-pairs, in addition to or in parallel with genetic analyses that were performed during the diagnostic work-up of the study participants., Results: In ten out of these 35 families (29 %), we found mutations in genes already known to be related to a disorder in which microcephaly is a main feature. Two unrelated patients had mutations in the ASPM gene. In seven other patients we found mutations in RAB3GAP1, RNASEH2B, KIF11, ERCC8, CASK, DYRK1A and BRCA2. In one of the sib-pairs, mutations were found in the RTTN gene. Mutations were present in seven out of our ten families with an established etiological diagnosis with recessive inheritance., Conclusions: We demonstrate that whole-exome sequencing is a powerful tool for the diagnostic evaluation of patients with highly heterogeneous neurodevelopmental disorders such as intellectual disability with microcephaly. Our results confirm that autosomal recessive disorders are highly prevalent among patients with microcephaly.

Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function., (Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Familial glucocorticoid deficiency (FGD) is an autosomal recessive disorder characterized by low levels of cortisol despite high adrenocorticotropin (ACTH) levels, due to the reduced ability of the adrenal cortex to produce cortisol in response to stimulation by ACTH. FGD is a heterogeneous disorder for which causal mutations have been identified in MC2R, MRAP, MCM4 and TXNRD2. Also mutations in STAR and CYP11A1 can sometimes present with a phenotype resembling FGD. Recently, it has been indicated that FGD can also be caused by mutations in NNT (nicotinamide nucleotide transhydrogenase). We identified a 6.67 Mb homozygous region harboring the NNT gene by SNP haplotyping in a 1-year old Dutch boy presenting with FGD, but without mutations in MC2R and MRAP. Exome-sequencing revealed a novel homozygous mutation (NM&#95;012343.3: c.1259dupG) in NNT that was predicted to be disease-causing. The mutation is located in exon 9 and creates a frameshift leading to a premature stop-codon (p.His421Serfs*4) that is known to result in FGD. Both parents were shown to be heterozygous carriers. We reviewed the literature for all the reported NNT mutations and their clinical presentation. The median age of disease onset in 23 reported patients, including the present patient, was 12 months (range 3 days-39 months). There was no difference in age of disease onset between truncating and non-truncating NNT mutations. Based on recent literature, we advise to monitor patients with FGD due to NNT mutations for possible combined mineralocorticoid insufficiency and extra-adrenal manifestations., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

CHARGE syndrome is a variable, multiple congenital malformation syndrome. Patients with CHARGE syndrome have frequent infections that are presumed to be due to anatomical anomalies of the craniofacial region and upper airway, and cranial nerve problems resulting in swallowing difficulties and aspiration. The possible contribution of immunological abnormalities to these infections has not been systematically studied even though immune deficiencies have been described in patients with 22q11.2 deletion syndrome, a condition which shares remarkable clinical overlap with CHARGE syndrome. We assessed the frequency and nature of immune dysfunction in 24 children with genetically proven CHARGE syndrome. All patients, or their parents, completed a questionnaire on infectious history. Their immune system was extensively assessed through full blood counts, immunoglobulin levels, lymphocyte subpopulations, peripheral B- and T-cell differentiation, T-receptor excision circle (TREC) analysis, T-cell function, and vaccination responses. All CHARGE patients had a history of infections (often frequent), mainly otitis media and pneumonia, leading to frequent use of antibiotics and to hospital admissions. Decreased T-cell numbers were found in 12 (50%) patients, presumably caused by insufficient thymic output since TREC amounts were also diminished in CHARGE patients. Despite normal peripheral B-cell differentiation and immunoglobulin production in all patients, 83% of patients had insufficient antibody titers to one or more early childhood vaccinations. Based on our results, we recommend immunological evaluation of CHARGE patients with recurrent infections.

CHARGE syndrome is caused by a dominant variant in the CHD7 gene. Multiple organ systems can be affected because of haploinsufficiency of CHD7 during embryonic development. CHARGE syndrome shares many clinical features with the 22q11.2 deletion syndrome. Immunological abnormalities have been described, but are generally given little attention in studies on CHARGE syndrome. However, structured information on immunological abnormalities in CHARGE patients is necessary to develop optimal guidelines for diagnosis, treatment and follow-up in these patients. Here, we provide an overview of the current literature on immunological abnormalities in CHARGE syndrome. We also explore immunological abnormalities in comparable multiple congenital anomaly syndromes to identify common immunological phenotypes and genetic pathways that might regulate the immune system. Finally, we aim to identify gaps in our knowledge on the immunological aspects in CHARGE syndrome that need further study.

Interstitial deletions of chromosome 3p14p12 are a rare chromosome rearrangement. Twenty-six patients have been reported in the literature to date, however, a specific clinical phenotype has not yet been delineated. We describe three patients (two new) with overlapping chromosome 3p14p12 deletions and review the clinical and molecular data of 11 well-characterized, published cases. These patients had a number of features in common, such as short stature, failure to thrive, facial dysmorphism, congenital heart defects, urogenital abnormalities, neurological problems, hearing loss, and global developmental delay, suggesting that the interstitial chromosome 3p14p12 deletion gives rise to a multiple congenital anomaly syndrome. Some of the patients show clinical overlap with other complex syndromes such as CHARGE syndrome. Genotype-phenotype analysis revealed candidate genes for parts of the clinical features suggesting that the 3p14 deletion is a contiguous gene syndrome., (© 2015 Wiley Periodicals, Inc.)

Study Question: Do clinical characteristics of recurrent miscarriage couples with a chromosomal abnormality and who opt for PGD differ from couples that decline PGD after extensive genetic counselling?, Summary Answer: No differences in clinical characteristics are identified between recurrent miscarriage couples carrying a structural chromosomal abnormality who opt for PGD compared with those that decline PGD after extensive genetic counselling., What Is Known Already: Couples who have experienced two or more miscarriages (recurrent miscarriage) are at increased recurrence risk if one of the partners carries a structural chromosomal abnormality. PGD can be offered to avoid (another) miscarriage or pregnancy termination when (invasive) prenatal diagnosis shows an abnormal result. To date, no reports are available that describe reproductive decision-making after genetic counselling on PGD in these specific couples., Study Design, Size, Duration: Retrospective cohort study of 294 couples carrying a structural chromosomal abnormality seeking genetic counselling on PGD between 1996 and 2012., Participants/materials, Setting, Methods: Participants were recurrent miscarriage couples carrying a structural chromosomal abnormality. They had been referred for genetic counselling to the only national licensed PGD centre. Clinical characteristics analysed included couple associated characteristics, characteristics concerning reproductive history and external characteristics such as type of physician that referred the couple for genetic counselling and the clinical geneticist performing the counselling on PGD., Main Results and the Role of Chance: Of 294 couples referred for counselling on PGD, 26 were not accepted because they did not meet the criteria for IVF-PGD. The remaining cohort of 268 couples consisted of two-thirds female and one-third male carriers. Main PGD indications were reciprocal translocations (83.9%) and Robertsonian translocations (16.7%). Following genetic counselling, 76.9% of included couples chose PGD as their reproductive option, the others declined PGD. Reproductive choice is not influenced by sex of the translocation carrier (P = 0.499), type of chromosomal abnormality (P = 0.346), number of previous miscarriages (P = 0.882), history of termination of pregnancy (TOP) because of an unbalanced fetal karyotype (P = 0.800), referring physician (P = 0.208) or geneticist who performed the counselling (P = 0.410)., Limitations, Reasons for Caution: This study only included recurrent miscarriage couples carrying a structural chromosomal abnormality, who were actually referred to a PGD clinic for genetic counselling. We lack information on couples who were not referred for PGD. Some of these patients may not have been informed on PGD at all, while others were not referred for counselling because they did not opt for PGD to start with., Wider Implications of the Findings: This study shows that reproductive choices in couples with recurrent miscarriage on the basis of a structural chromosomal abnormality are not influenced by characteristics of the couple itself, nor by their obstetric history or external characteristics. These findings suggest that a couples' intrinsic attitude towards PGD treatment is a major factor influencing their reproductive choice. Future research will focus on these personal motives that seem to push reproductive decision-making following genetic counselling in a given direction., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Since 2004, CHD7 mutations have been a known cause of CHARGE (Coloboma, Heart defects, Atresia of choane, Retardation of growth and development, Genital hypoplasia, Ear anomalies) syndrome, but the full clinical spectrum of CHD7 mutations is only now gradually emerging. CHD7 mutations have been identified in patients who do not fulfill the clinical criteria for CHARGE syndrome and in patients with overlapping syndromes. Variable congenital heart defects occur in the majority of patients with CHD7 mutations, with an overrepresentation of atrioventricular septal defects and conotruncal heart defects. This prompted us to study CHD7 in 46 patients with these heart defects and one other feature of CHARGE syndrome. We identified two CHD7 variants that were inherited from a healthy parent (c.3778 + 17C > T, c.7294G > A), but no pathogenic CHD7 mutations. We conclude that CHD7 mutations are not a major cause of the atrioventricular septal defects and conotruncal heart defects, not even if one extra phenotypic feature of CHARGE syndrome is present. Therefore, CHD7 analysis should not be performed routinely in this group of patients. However, we do recommend adding CHD7 to massive parallel sequencing gene panels for diagnostic work in patients with syndromic heart defects., (© 2014 Wiley Periodicals, Inc.)

Microdeletions of the 5q11.2 region are rare; in literature only two patients with a deletion in this region have been reported so far. In this study, we describe four additional patients and further define this new 5q11.2 microdeletion syndrome. A comparison of the features observed in all six patients with overlapping 5q11.2 deletions showed a phenotypic spectrum that overlaps with CHARGE syndrome and 22q11.2 deletion syndrome including choanal atresia, developmental delay, heart defects, external ear abnormalities, and short stature. No colobomas or abnormalities of semicircular canals and olfactory nerves were reported. Two male patients had genital abnormalities. We estimated a 2.0 Mb (53.0-55.0 Mb) Shortest Region of Overlap (SRO) for the main clinical characteristics of the syndrome. This region contains nine genes and two non-coding microRNAs. In this region DHX29 serves as the candidate gene as it encodes an ATP-dependent RNA-helicase that is involved in the initiation of RNA translation. Screening a small cohort of 14 patients who presented the main features, however, did not reveal any pathogenic abnormalities of DHX29., (© 2014 Wiley Periodicals, Inc.)

22q11.2 deletion syndrome is one of the most common microdeletion syndromes. Most patients have a deletion resulting from a recombination of low copy repeat blocks LCR22-A and LCR22-D. Loss of the TBX1 gene is considered the most important cause of the phenotype. A limited number of patients with smaller, overlapping deletions distal to the TBX1 locus have been described in the literature. In these patients, the CRKL gene is deleted. Haploinsufficiency of this gene has also been implicated in the pathogenesis of 22q11.2 deletion syndrome. To distinguish these deletions (comprising the LCR22-B to LCR22-D region) from the more distal 22q11.2 deletions (located beyond LCR22-D), we propose the term "central 22q11.2 deletions". In the present study we report on 27 new patients with such a deletion. Together with information on previously published cases, we review the clinical findings of 52 patients. The prevalence of congenital heart anomalies and the frequency of de novo deletions in patients with a central deletion are substantially lower than in patients with a common or distal 22q11.2 deletion. Renal and urinary tract malformations, developmental delays, cognitive impairments and behavioral problems seem to be equally frequent as in patients with a common deletion. None of the patients had a cleft palate. Patients with a deletion that also encompassed the MAPK1 gene, located just distal to LCR22-D, have a different and more severe phenotype, characterized by a higher prevalence of congenital heart anomalies, growth restriction and microcephaly. Our results further elucidate genotype-phenotype correlations in 22q11.2 deletion syndrome spectrum., (© 2014 Wiley Periodicals, Inc.)

Heterozygous loss of function mutations in CHD7 (chromodomain helicase DNA-binding protein 7) lead to CHARGE syndrome, a complex developmental disorder affecting craniofacial structures, cranial nerves and several organ systems. Recently, it was demonstrated that CHD7 is essential for the formation of multipotent migratory neural crest cells, which migrate from the neural tube to many regions of the embryo, where they differentiate into various tissues including craniofacial and heart structures. So far, only few CHD7 target genes involved in neural crest cell development have been identified and the role of CHD7 in neural crest cell guidance and the regulation of mesenchymal-epithelial transition are unknown. Therefore, we undertook a genome-wide microarray expression analysis on wild-type and CHD7 deficient (Chd7 (Whi/+) and Chd7 (Whi/Whi)) mouse embryos at day 9.5, a time point of neural crest cell migration. We identified 98 differentially expressed genes between wild-type and Chd7 (Whi/Whi) embryos. Interestingly, many misregulated genes are involved in neural crest cell and axon guidance such as semaphorins and ephrin receptors. By performing knockdown experiments for Chd7 in Xenopus laevis embryos, we found abnormalities in the expression pattern of Sema3a, a protein involved in the pathogenesis of Kallmann syndrome, in vivo. In addition, we detected non-synonymous SEMA3A variations in 3 out of 45 CHD7-negative CHARGE patients. In summary, we discovered for the first time that Chd7 regulates genes involved in neural crest cell guidance, demonstrating a new aspect in the pathogenesis of CHARGE syndrome. Furthermore, we showed for Sema3a a conserved regulatory mechanism across different species, highlighting its significance during development. Although we postulated that the non-synonymous SEMA3A variants which we found in CHD7-negative CHARGE patients alone are not sufficient to produce the phenotype, we suggest an important modifier role for SEMA3A in the pathogenesis of this multiple malformation syndrome.

Mutations in CHD7 are the major cause of CHARGE syndrome, an autosomal dominant disorder with an estimated prevalence of 1/15,000. We have little understanding of the disruptions in the developmental programme that underpin brain defects associated with this syndrome. Using mouse models, we show that Chd7 haploinsufficiency results in reduced Fgf8 expression in the isthmus organiser (IsO), an embryonic signalling centre that directs early cerebellar development. Consistent with this observation, Chd7 and Fgf8 loss-of-function alleles interact during cerebellar development. CHD7 associates with Otx2 and Gbx2 regulatory elements and altered expression of these homeobox genes implicates CHD7 in the maintenance of cerebellar identity during embryogenesis. Finally, we report cerebellar vermis hypoplasia in 35% of CHARGE syndrome patients with a proven CHD7 mutation. These observations provide key insights into the molecular aetiology of cerebellar defects in CHARGE syndrome and link reduced FGF signalling to cerebellar vermis hypoplasia in a human syndrome. DOI: http://dx.doi.org/10.7554/eLife.01305.001.

Unlabelled: Preimplantation genetic diagnosis (PGD) is offered to couples carrying a reciprocal translocation in an attempt to increase their chance of phenotypically normal offspring. For the selection of embryos that are balanced for the translocation chromosomes, it is critical to use a combination of DNA probes that can take account of all the segregation patterns of the particular translocation. The frequency of the different segregation types differs depending on the chromosomes involved, the location of the breakpoints and the number of chiasmata and the sex of the carrier. We report on a case of misdiagnosis after PGD-fluorescence in situ hybridization in a female translocation 46,X,t(X;5)(q13;p14) carrier. Transfer of two embryos diagnosed as balanced for the translocation chromosomes resulted in a singleton pregnancy that miscarried at 8 weeks' gestational age. The unbalanced karyotype of the fetus was consistent with 3:1 segregation resulting in tertiary trisomy for the derivative chromosome 5: 47,XX,+der(5)t(X;5)(q13;p14)mat. Based on additional molecular cytogenetic studies of fetal tissue and the initially investigated blastomeres, we concluded that the misdiagnosis was most probably due to a technical error, i.e. a partial hybridization failure or co-localization of the Xq/Yq subtelomere probe signals. No evidence for a normal cell line (mosaicism) was found in the fetus, which could have explained the discrepancy. This case demonstrates the importance of using two diagnostic probes or testing 2 cells to detect translocation products with potentially viable imbalance. X;autosome translocations are a special case due to the added complication of X chromosome inactivation and particular caution is advised when designing a PGD strategy., Trial Registration Number: not applicable.

The spindle assembly checkpoint controls proper chromosome segregation during mitosis and prevents aneuploidy-an important feature of cancer cells. We performed genome-wide and targeted copy number and mutation analyses of germline DNA from 208 patients with familial or early-onset (40 years of age or younger) colorectal cancer; we identified haploinsufficiency or heterozygous mutations in the spindle assembly checkpoint genes BUB1 and BUB3 in 2.9% of them. Besides colorectal cancer, these patients had variegated aneuploidies in multiple tissues and variable dysmorphic features. These results indicate that mutations in BUB1 and BUB3 cause mosaic variegated aneuploidy and increase the risk of colorectal cancer at a young age., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Otocephaly-dysgnathia complex is characterized by mandibular hypo- or aplasia, ear abnormalities, microstomia, and microglossia. Mutations in the orthodenticle homeobox 2 (OTX2) and paired related homeobox 1 (PRRX1) genes have recently been identified in some cases. We screened 4 otocephalic cases for these 2 genes and identified OTX2 mutations in 2 of them, thus confirming OTX2 is implicated in otocephaly. No PRRX1 mutation was identified. Interestingly, ocular involvement is not a constant feature in otocephalic cases with an OTX2 mutation. In one case, the mutation was inherited from a microphthalmic mother. The mechanism underlying this intrafamilial phenotypic variability remains unclear, but other genetic factors are likely to be necessary for the manifestation of the otocephalic phenotype.

CHARGE (coloboma, heart defects, atresia of choanae, retardation of growth and development, genital hypoplasia, and ear abnormalities) and 22q11.2 deletion syndromes are variable, congenital malformation syndromes that show considerable phenotypic overlap. We further explored this clinical overlap and proposed recommendations for the genetic diagnosis of both syndromes. We described 2 patients clinically diagnosed with CHARGE syndrome, who were found to carry a 22q11.2 deletion, and searched the literature for more cases. In addition, we screened our cohort of CHD7 mutation carriers (n = 802) for typical 22q11.2 deletion features and studied CHD7 in 20 patients with phenotypically 22q11.2 deletion syndrome but without haploinsufficiency of TBX1. In total, we identified 5 patients with a clinical diagnosis of CHARGE syndrome and a proven 22q11.2 deletion. Typical 22q11.2 deletion features were found in 30 patients (30/802, 3.7%) of our CHD7 mutation-positive cohort. We found truncating CHD7 mutations in 5/20 patients with phenotypically 22q11.2 deletion syndrome. Differentiating between CHARGE and 22q11.2 deletion syndromes can be challenging. CHD7 and TBX1 probably share a molecular pathway or have common target genes in affected organs. We strongly recommend performing CHD7 analysis in patients with a 22q11.2 deletion phenotype without TBX1 haploinsufficiency and conversely, performing a genome-wide array in CHARGE syndrome patients without a CHD7 mutation.

Background: Loss-of-function mutations in CHD7 cause Coloboma, Heart Disease, Atresia of Choanae, Retardation of Growth and/or Development, Genital Hypoplasia, and Ear Abnormalities With or Without Deafness (CHARGE) syndrome, a variable combination of multiple congenital malformations including heart defects. Heart defects are reported in 70% to 92% of patients with a CHD7 mutation, but most studies are small and do not provide a detailed classification of the defects. We present the first, detailed, descriptive study on the cardiac phenotype of 299 patients with a CHD7 mutation and discuss the role of CHD7 in cardiac development., Methods and Results: We collected information on congenital heart defects in 299 patients with a pathogenic CHD7 mutation, of whom 220 (74%) had a congenital heart defect. Detailed information on the heart defects was available for 202 of these patients. We classified the heart defects based on embryonic cardiac development and compared the distribution to 1007 equally classified nonsyndromic heart defects of patients registered by EUROCAT, a European Registry of Congenital Anomalies. Heart defects are highly variable in patients with CHD7 mutations, but atrioventricular septal defects and conotruncal heart defects are over-represented. Sex did not have an effect on the presence of heart defects, but truncating CHD7 mutations resulted in a heart defect significantly more often than missense or splice-site mutations (χ², P<0.001)., Conclusions: CHD7 plays an important role in cardiac development, given that we found a wide range of heart defects in 74% of a large cohort of patients with a CHD7 mutation. Conotruncal defects and atrioventricular septal defects are over-represented in patients with CHD7 mutations compared with patients with nonsyndromic heart defects.

We previously reported on nonrecurrent overlapping duplications at Xp11.22 in individuals with nonsyndromic intellectual disability (ID) harboring HSD17B10, HUWE1, and the microRNAs miR-98 and let-7f-2 in the smallest region of overlap. Here, we describe six additional individuals with nonsyndromic ID and overlapping microduplications that segregate in the families. High-resolution mapping of the 12 copy-number gains reduced the minimal duplicated region to the HUWE1 locus only. Consequently, increased mRNA levels were detected for HUWE1, but not HSD17B10. Marker and SNP analysis, together with identification of two de novo events, suggested a paternally derived intrachromosomal duplication event. In four independent families, we report on a polymorphic 70 kb recurrent copy-number gain, which harbors part of HUWE1 (exon 28 to 3' untranslated region), including miR-98 and let-7f-2. Our findings thus demonstrate that HUWE1 is the only remaining dosage-sensitive gene associated with the ID phenotype. Junction and in silico analysis of breakpoint regions demonstrated simple microhomology-mediated rearrangements suggestive of replication-based duplication events. Intriguingly, in a single family, the duplication was generated through nonallelic homologous recombination (NAHR) with the use of HUWE1-flanking imperfect low-copy repeats, which drive this infrequent NAHR event. The recurrent partial HUWE1 copy-number gain was also generated through NAHR, but here, the homologous sequences used were identified as TcMAR-Tigger DNA elements, a template that has not yet been reported for NAHR. In summary, we showed that an increased dosage of HUWE1 causes nonsyndromic ID and demonstrated that the Xp11.22 region is prone to recombination- and replication-based rearrangements., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

CHD7 is a member of the chromodomain helicase DNA-binding (CHD) protein family that plays a role in transcription regulation by chromatin remodeling. Loss-of-function mutations in CHD7 are known to cause CHARGE syndrome, an autosomal-dominant malformation syndrome in which several organ systems, for example, the central nervous system, eye, ear, nose, and mediastinal organs, are variably involved. In this article, we review all the currently described CHD7 variants, including 183 new pathogenic mutations found by our laboratories. In total, we compiled 528 different pathogenic CHD7 alterations from 508 previously published patients with CHARGE syndrome and 294 unpublished patients analyzed by our laboratories. The mutations are equally distributed along the coding region of CHD7 and most are nonsense or frameshift mutations. Most mutations are unique, but we identified 94 recurrent mutations, predominantly arginine to stop codon mutations. We built a locus-specific database listing all the variants that is easily accessible at www.CHD7.org. In addition, we summarize the latest data on CHD7 expression studies, animal models, and functional studies, and we discuss the latest clinical insights into CHARGE syndrome., (© 2012 Wiley Periodicals, Inc.)

CHARGE syndrome is characterized by the variable occurrence of multisensory impairment, congenital anomalies, and developmental delay, and is caused by heterozygous mutations in the CHD7 gene. Correct interpretation of CHD7 variants is essential for genetic counseling. This is particularly difficult for missense variants because most variants in the CHD7 gene are private and a functional assay is not yet available. We have therefore developed a novel classification system to predict the pathogenic effects of CHD7 missense variants that can be used in a diagnostic setting. Our classification system combines the results from two computational algorithms (PolyPhen-2 and Align-GVGD) and the prediction of a newly developed structural model of the chromo- and helicase domains of CHD7 with segregation and phenotypic data. The combination of different variables will lead to a more confident prediction of pathogenicity than was previously possible. We have used our system to classify 145 CHD7 missense variants. Our data show that pathogenic missense mutations are mainly present in the middle of the CHD7 gene, whereas benign variants are mainly clustered in the 5' and 3' regions. Finally, we show that CHD7 missense mutations are, in general, associated with a milder phenotype than truncating mutations., (© 2012 Wiley Periodicals, Inc.)

The range of commercially available array platforms and analysis software packages is expanding and their utility is improving, making reliable detection of copy-number variants (CNVs) relatively straightforward. Reliable interpretation of CNV data, however, is often difficult and requires expertise. With our knowledge of the human genome growing rapidly, applications for array testing continuously broadening, and the resolution of CNV detection increasing, this leads to great complexity in interpreting what can be daunting data. Correct CNV interpretation and optimal use of the genotype information provided by single-nucleotide polymorphism probes on an array depends largely on knowledge present in various resources. In addition to the availability of host laboratories' own datasets and national registries, there are several public databases and Internet resources with genotype and phenotype information that can be used for array data interpretation. With so many resources now available, it is important to know which are fit-for-purpose in a diagnostic setting. We summarize the characteristics of the most commonly used Internet databases and resources, and propose a general data interpretation strategy that can be used for comparative hybridization, comparative intensity, and genotype-based array data., Competing Interests: Statement: The authors declare no conflict of interest.

Genome-wide arrays are rapidly replacing conventional karyotyping in postnatal cytogenetic diagnostics and there is a growing request for arrays in the prenatal setting. Several studies have documented 1-3% additional abnormal findings in prenatal diagnosis with arrays compared to conventional karyotyping. A recent meta-analysis demonstrated that 5.2% extra diagnoses can be expected in fetuses with ultrasound abnormalities. However, no consensus exists as to whether the use of genome-wide arrays should be restricted to pregnancies with ultrasound abnormalities, performed in all women undergoing invasive prenatal testing or offered to all pregnant women. Moreover, the interpretation of array results in the prenatal situation is challenging due to the large numbers of copy number variants with no major phenotypic effect. This also raises the question of what, or what not to report, for example, how to deal with unsolicited findings. These issues were discussed at a working group meeting that preceded the European Society of Human Genetics 2011 Conference in Amsterdam. This article is the result of this meeting and explores the introduction of genome-wide arrays into routine prenatal diagnosis. We aim to give some general recommendations on how to develop practical guidelines that can be implemented in the local setting and that are consistent with the emerging international consensus., (© 2012 Wiley Periodicals, Inc.)

Context: Kallmann syndrome (KS) and CHARGE syndrome are rare heritable disorders in which anosmia and hypogonadotropic hypogonadism co-occur. KS is genetically heterogeneous, and there are at least eight genes involved in its pathogenesis, whereas CHARGE syndrome is caused by autosomal dominant mutations in only one gene, the CHD7 gene. Two independent studies showed that CHD7 mutations can also be found in a minority of KS patients., Objective: We aimed to investigate whether CHD7 mutations can give rise to isolated KS or whether additional features of CHARGE syndrome always occur., Design: We performed CHD7 analysis in a cohort of 36 clinically well-characterized Dutch patients with KS but without mutations in KAL1 and with known status for the KS genes with incomplete penetrance, FGFR1, PROK2, PROKR2, and FGF8., Results: We identified three heterozygous CHD7 mutations. The CHD7-positive patients were carefully reexamined and were all found to have additional features of CHARGE syndrome., Conclusion: The yield of CHD7 analysis in patients with isolated KS seems very low but increases when additional CHARGE features are present. Therefore, we recommend performing CHD7 analysis in KS patients who have at least two additional CHARGE features or semicircular canal anomalies. Identifying a CHD7 mutation has important clinical implications for the surveillance and genetic counseling of patients.

Kleefstra syndrome is characterized by the core phenotype of developmental delay/intellectual disability, (childhood) hypotonia and distinct facial features. The syndrome can be either caused by a microdeletion in chromosomal region 9q34.3 or by a mutation in the euchromatin histone methyltransferase 1 (EHMT1) gene. Since the early 1990s, 85 patients have been described, of which the majority had a 9q34.3 microdeletion (>85%). So far, no clear genotype-phenotype correlation could be observed by studying the clinical and molecular features of both 9q34.3 microdeletion patients and patients with an intragenic EHMT1 mutation. Thus, to further expand the genotypic and phenotypic knowledge about the syndrome, we here report 29 newly diagnosed patients, including 16 patients with a 9q34.3 microdeletion and 13 patients with an EHMT1 mutation, and review previous literature. The present findings are comparable to previous reports. In addition to our former findings and recommendations, we suggest cardiac screening during follow-up, because of the possible occurrence of cardiac arrhythmias. In addition, clinicians and caretakers should be aware of the regressive behavioral phenotype that might develop at adolescent/adult age and seems to have no clear neurological substrate, but is rather a so far unexplained neuropsychiatric feature.

Brain malformations are individually rare but collectively common causes of developmental disabilities. Many forms of malformation occur sporadically and are associated with reduced reproductive fitness, pointing to a causative role for de novo mutations. Here, we report a study of Baraitser-Winter syndrome, a well-defined disorder characterized by distinct craniofacial features, ocular colobomata and neuronal migration defect. Using whole-exome sequencing of three proband-parent trios, we identified de novo missense changes in the cytoplasmic actin-encoding genes ACTB and ACTG1 in one and two probands, respectively. Sequencing of both genes in 15 additional affected individuals identified disease-causing mutations in all probands, including two recurrent de novo alterations (ACTB, encoding p.Arg196His, and ACTG1, encoding p.Ser155Phe). Our results confirm that trio-based exome sequencing is a powerful approach to discover genes causing sporadic developmental disorders, emphasize the overlapping roles of cytoplasmic actin proteins in development and suggest that Baraitser-Winter syndrome is the predominant phenotype associated with mutation of these two genes.

The correct interpretation of copy number gains in patients with developmental delay and multiple congenital anomalies is hampered by the large number of copy number variations (CNVs) encountered in healthy individuals. The variable phenotype associated with copy number gains makes interpretation even more difficult. Literature shows that inheritence, size and presence in healthy individuals are commonly used to decide whether a certain copy number gain is pathogenic, but no general consensus has been established. We aimed to develop guidelines for interpreting gains detected by array analysis using array CGH data of 300 patients analysed with the 105K Agilent oligo array in a diagnostic setting. We evaluated the guidelines in a second, independent, cohort of 300 patients. In the first 300 patients 797 gains of four or more adjacent oligonucleotides were observed. Of these, 45.4% were de novo and 54.6% were familial. In total, 94.8% of all de novo gains and 87.1% of all familial gains were concluded to be benign CNVs. Clinically relevant gains ranged from 288 to 7912 kb in size, and were significantly larger than benign gains and gains of unknown clinical relevance (P < 0.001). Our study showed that a threshold of 200 kb is acceptable in a clinical setting, whereas heritability does not exclude a pathogenic nature of a gain. Evaluation of the guidelines in the second cohort of 300 patients revealed that the interpretation guidelines were clear, easy to follow and efficient.

Congenital aural atresia (CAA) can occur as an isolated congenital malformation or in the context of a number of monogenic and chromosomal syndromes. CAA is frequently seen in individuals with an 18q deletion, which is characterized by intellectual disability, reduced white-matter myelination, foot deformities, and distinctive facial features. Previous work has indicated that a critical region for CAA is located in 18q22.3. We studied four individuals (from two families) with CAA and other features suggestive of an 18q deletion, and we detected overlapping microdeletions in 18q22.3 in both families. The minimal region of deletion overlap (72.9-73.4 Mb) contained only one known gene, TSHZ1, which was recently shown to be important for murine middle-ear development. Sequence analysis of the coding exons in TSHZ1 in a cohort of 11 individuals with isolated, nonsyndromic bilateral CAA revealed two mutations, c.723G>A (p.Trp241X) and c.946&#95;947delinsA (p.Pro316ThrfsX16), and both mutations predicted a loss of function. Together, these results demonstrate that hemizygosity of TSHZ1 leads to congenital aural atresia as a result of haploinsufficiency., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

A partial deletion of chromosome band 2p25.3 (2pter) is a rarely described cytogenetic aberration in patients with intellectual disability (ID). Using microarrays we identified deletions of 2p25.3, sized 0.37-3.13 Mb, in three adult siblings and three unrelated patients. All patients had ID, obesity or overweight and/or a square-shaped stature without overt facial dysmorphic features. Combining our data with phenotypic and genotypic data of three patients from the literature we defined the minimal region of overlap which contained one gene, i.e., MYT1L. MYT1L is highly transcribed in the mouse embryonic brain where its expression is restricted to postmitotic differentiating neurons. In mouse-induced pluripotent stem cell (iPS) models, MYT1L is essential for inducing functional mature neurons. These resemble excitatory cortical neurons of the forebrain, suggesting a role for MYT1L in development of cognitive functions. Furthermore, MYT1L can directly convert human fibroblasts into functional neurons in conjunction with other transcription factors. MYT1L duplication was previously reported in schizophrenia, indicating that the gene is dosage-sensitive and that shared neurodevelopmental pathways may be affected in ID and schizophrenia. Finally, deletion of MYT1, another member of the Myelin Transcription Factor family involved in neurogenesis and highly similar to MYT1L, was recently described in ID as well. The identification of MYT1L as candidate gene for ID justifies further molecular studies aimed at detecting mutations and for mechanistic studies on its role in neuron development and on neuropathogenic effects of haploinsufficiency., (Copyright © 2011 Wiley Periodicals, Inc.)