De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females.

Variants in CLCN4, which encodes the chloride/hydrogen ion exchanger CIC-4 prominently expressed in brain, were recently described to cause X-linked intellectual disability and epilepsy. We present detailed phenotypic information on 52 individuals from 16 families with CLCN4-related disorder: 5 affected females and 2 affected males with a de novo variant in CLCN4 (6 individuals previously unreported) and 27 affected males, 3 affected females and 15 asymptomatic female carriers from 9 families with inherited CLCN4 variants (4 families previously unreported). Intellectual disability ranged from borderline to profound. Behavioral and psychiatric disorders were common in both child- and adulthood, and included autistic features, mood disorders, obsessive-compulsive behaviors and hetero- and autoaggression. Epilepsy was common, with severity ranging from epileptic encephalopathy to well-controlled seizures. Several affected individuals showed white matter changes on cerebral neuroimaging and progressive neurological symptoms, including movement disorders and spasticity. Heterozygous females can be as severely affected as males. The variability of symptoms in females is not correlated with the X inactivation pattern studied in their blood. The mutation spectrum includes frameshift, missense and splice site variants and one single-exon deletion. All missense variants were predicted to affect CLCN4's function based on in silico tools and either segregated with the phenotype in the family or were de novo. Pathogenicity of all previously unreported missense variants was further supported by electrophysiological studies in Xenopus laevis oocytes. We compare CLCN4-related disorder with conditions related to dysfunction of other members of the CLC family.

The Cognitive and Behavioral Phenotypes of Individuals with CHRNA7 Duplications.

Chromosome 15q11q13 is among the least stable regions in the genome due to its highly complex genomic architecture. Low copy repeat elements at 15q13.3 facilitate recurrent copy number variants (CNVs), with deletions established as pathogenic and CHRNA7 implicated as a candidate gene. However, the pathogenicity of duplications of CHRNA7 is unclear, as they are found in affected probands as well as in reportedly healthy parents and unaffected control individuals. We evaluated 18 children with microduplications involving CHRNA7, identified by clinical chromosome microarray analysis (CMA). Comprehensive phenotyping revealed high prevalence of developmental delay/intellectual disability, autism spectrum disorder, and attention deficit/hyperactivity disorder. As CHRNA7 duplications are the most common CNVs identified by clinical CMA, this study provides anticipatory guidance for those involved with care of affected individuals.

Microduplication of Xp11.23p11.3 with effects on cognition, behavior, and craniofacial development.

We report an ~1.3 Mb tandem duplication at Xp11.23p11.3 in an 11-year-old boy with pleasant personality, hyperactivity, learning and visual-spatial difficulties, relative microcephaly, long face, stellate iris pattern, and periorbital fullness. This clinical presentation is milder and distinct from that of patients with partially overlapping Xp11.22p11.23 duplications which have been described in males and females with intellectual disability, language delay, autistic behaviors, and seizures. The duplicated region harbors three known X-linked mental retardation genes: FTSJ1, ZNF81, and SYN1. Quantitative polymerase chain reaction from whole blood total RNA showed increased expression of three genes located in the duplicated region: EBP, WDR13, and ZNF81. Thus, over-expression of genes in the interval may contribute to the observed phenotype. Many of the features seen in this patient are present in individuals with Williams-Beuren syndrome (WBS). Interestingly, the SYN1 gene within the duplicated interval, as well as the STX1A gene, within the WBS critical region, co-localize to presynaptic active zones, and play important roles in neurotransmitter release.

22q13.3 deletion syndrome: clinical and molecular analysis using array CGH.

The 22q13.3 deletion syndrome results from loss of terminal segments of varying sizes at 22qter. Few genotype-phenotype correlations have been found but all patients have mental retardation and severe delay, or absence of, expressive speech. We carried out clinical and molecular characterization of 13 patients. Developmental delay and speech abnormalities were common to all and comparable in frequency and severity to previously reported cases. Array-based comparative genomic hybridization showed the deletions to vary from 95 kb to 8.5 Mb. We also carried out high-resolution 244K array comparative genomic hybridization in 10 of 13 patients, that defined the proximal and distal breakpoints of each deletion and helped determine the size, extent, and gene content within the deletion. Two patients had a smaller 95 kb terminal deletion with breakpoints within the SHANK3 gene while three other patients had a similar 5.5 Mb deletion implying the recurrent nature of these deletions. The two largest deletions were found in patients with ring chromosome 22. No correlation could be made with deletion size and phenotype although complete/partial SHANK3 was deleted in all patients. There are very few reports on array comparative genomic hybridization analysis on patients with the 22q13.3 deletion syndrome, and we aim to accurately characterize these patients both clinically and at the molecular level, to pave the way for further genotype-phenotype correlations. (c) 2010 Wiley-Liss, Inc.

Microdeletions including YWHAE in the Miller-Dieker syndrome region on chromosome 17p13.3 result in facial dysmorphisms, growth restriction, and cognitive impairment.

BACKGROUND: Deletions in the 17p13.3 region are associated with abnormal neuronal migration. Point mutations or deletion copy number variants of the PAFAH1B1 gene in this genomic region cause lissencephaly, whereas extended deletions involving both PAFAH1B1 and YWHAE result in Miller-Dieker syndrome characterised by facial dysmorphisms and a more severe grade of lissencephaly. The phenotypic consequences of YWHAE deletion without deletion of PAFAH1B1 have not been studied systematically. METHODS: We performed a detailed clinical and molecular characterization of five patients with deletions involving YWHAE but not PAFAH1B1, two with deletion including PAFAH1B1 but not YWHAE, and one with deletion of YWHAE and mosaic for deletion of PAFAH1B1. RESULTS: Three deletions were terminal whereas five were interstitial. Patients with deletions including YWHAE but not PAFAH1B1 presented with significant growth restriction, cognitive impairment, shared craniofacial features, and variable structural abnormalities of the brain. Growth restriction was not observed in one patient with deletion of YWHAE and TUSC5, implying that other genes in the region may have a role in regulation of growth with CRK being the most likely candidate. Using array based comparative genomic hybridisation and long range polymerase chain reaction, we have delineated the breakpoints of these nonrecurrent deletions and show that the interstitial genomic rearrangements are likely generated by diverse mechanisms, including the recently described Fork Stalling and Template Switching (FoSTeS)/Microhomology Mediated Break Induced Replication (MMBIR). CONCLUSIONS: Microdeletions of chromosome 17p13.3 involving YWHAE present with growth restriction, craniofacial dysmorphisms, structural abnormalities of brain and cognitive impairment. The interstitial deletions are mediated by diverse molecular mechanisms.

Microdeletion 15q13.3: a locus with incomplete penetrance for autism, mental retardation, and psychiatric disorders.

BACKGROUND: Microdeletions within chromosome 15q13.3 are associated both with a recently recognised syndrome of mental retardation, seizures, and dysmorphic features, and with schizophrenia. METHODS AND RESULTS: Based on routine diagnostic testing of approximately 8200 samples using array comparative genomic hybridisation, we identified 20 individuals (14 children and six parents in 12 families) with microdeletions of 15q13.3. Phenotypes in the children included developmental delay, mental retardation, or borderline IQ in most and autistic spectrum disorder (6/14), speech delay, aggressiveness, attention deficit hyperactivity disorder, and other behavioural problems. Both parents were available in seven families, and the deletion was de novo in one, inherited from an apparently normal parent in four, and inherited from a parent with learning disability and bipolar disorder in two families. Of the 14 children, six in five families were adopted, and DNA was available for only one of these 10 biological parents; the deletion was very likely inherited for one of these families with two affected children. Among the unavailable parents, two mothers were described as having mental retardation, another mother as having "mental illness", and one father as having schizophrenia. We hypothesise that some of the unavailable parents have the deletion. CONCLUSIONS: The occurrence of increased adoption, frequent autism, bipolar disorder, and lack of penetrance are noteworthy findings in individuals with deletion 15q13.3. A high rate of adoption may be related to the presence of the deletion in biological parents. Unconfirmed histories of antisocial behaviours in unavailable biological parents raise the concern that future research may show that deletion 15q13.3 is associated with such behaviours.

20p12.3 microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits.

BACKGROUND: Wolff-Parkinson-White syndrome (WPW) is a bypass re-entrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signalling in the development of annulus fibrosus in mice, it has been proposed that BMP signalling through the type 1a receptor and other downstream components may play a role in pre-excitation. METHODS AND RESULTS: Using the array comparative genomic hybridisation (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these harboured a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy number variant in the Database of Genomic Variants and has not been identified in 13 321 individuals from other cohort examined by array CGH in our laboratory. CONCLUSIONS: Our findings demonstrate a novel genomic disorder characterised by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.

Application of metaphase HR-CGH and targeted Chromosomal Microarray Analyses to genomic characterization of 116 patients with mental retardation and dysmorphic features.

Recent advances in molecular cytogenetics enable identification of small chromosomal aberrations that are undetectable by routine chromosome banding in 5-20% of patients with mental retardation/developmental delay (MR/DD) and dysmorphism. The aim of this study was to compare the clinical usefulness of two molecular cytogenetic techniques, metaphase high-resolution comparative genomic hybridization (HR-CGH) and targeted array CGH, also known as Chromosomal Microarray Analysis (CMA). A total of 116 patients with unexplained mild to severe MR and other features suggestive of a chromosomal abnormality with apparently normal or balanced karyotypes were analyzed using HR-CGH (43 patients) and/or CMA (91 patients). Metaphase HR-CGH detected seven interstitial deletions (16.3%). Rare deletions of chromosomes 16 (16p11.2p12.1) and 8 (8q21.11q21.2) were identified. Targeted CMA revealed copy-number changes in 19 of 91 patients (20.8%), among which 11 (11.8%) were clinically relevant, 6 (6.5%) were interpreted as polymorphic variants and 2 (2.1%) were of uncertain significance. The changes varied in size from 0.5 to 12.9 Mb. In summary, our results show that metaphase HR-CGH and array CGH techniques have become important components in cytogenetic diagnostics, particularly for detecting cryptic constitutional chromosome imbalances in patients with MR, in whom the underlying genetic defect is unknown. Additionally, application of both methods together increased the detection rates of genomic imbalances in the tested groups.

Different-sized duplications of Xq28, including MECP2, in three males with mental retardation, absent or delayed speech, and recurrent infections.

In XY males, duplication of any part of the X chromosome except the pseudoautosomal region leads to functional disomy of the corresponding genes. We describe three unrelated male patients with mental retardation (MR), absent or delayed speech, and recurrent infections. Using high-resolution comparative genomic hybridization (HR-CGH), whole genome array comparative genomic hybridization (array CGH), fluorescent in situ hybridization (FISH), and multiplex ligation probe amplification (MLPA), we have identified and characterized two different unbalanced Xq27.3-qter translocations on the Y chromosome (approx. 9 and 12 Mb in size) and one submicroscopic interstitial duplication (approx. 0.3-1.3 Mb) involving the MECP2 gene. Despite the differences in size of the duplicated segments, the patients share a clinical phenotype that overlaps with the features described in patients with MECP2 duplication. Our data confirm previous observations that MECP2 is the most important dosage-sensitive gene responsible for neurologic development in patients with duplications on the distal part of chromosome Xq.

Complex chromosome 17p rearrangements associated with low-copy repeats in two patients with congenital anomalies.

Recent molecular cytogenetic data have shown that the constitution of complex chromosome rearrangements (CCRs) may be more complicated than previously thought. The complicated nature of these rearrangements challenges the accurate delineation of the chromosomal breakpoints and mechanisms involved. Here, we report a molecular cytogenetic analysis of two patients with congenital anomalies and unbalanced de novo CCRs involving chromosome 17p using high-resolution array-based comparative genomic hybridization (array CGH) and fluorescent in situ hybridization (FISH). In the first patient, a 4-month-old boy with developmental delay, hypotonia, growth retardation, coronal synostosis, mild hypertelorism, and bilateral club feet, we found a duplication of the Charcot-Marie-Tooth disease type 1A and Smith-Magenis syndrome (SMS) chromosome regions, inverted insertion of the Miller-Dieker lissencephaly syndrome region into the SMS region, and two microdeletions including a terminal deletion of 17p. The latter, together with a duplication of 21q22.3-qter detected by array CGH, are likely the unbalanced product of a translocation t(17;21)(p13.3;q22.3). In the second patient, an 8-year-old girl with mental retardation, short stature, microcephaly and mild dysmorphic features, we identified four submicroscopic interspersed 17p duplications. All 17 breakpoints were examined in detail by FISH analysis. We found that four of the breakpoints mapped within known low-copy repeats (LCRs), including LCR17pA, middle SMS-REP/LCR17pB block, and LCR17pC. Our findings suggest that the LCR burden in proximal 17p may have stimulated the formation of these CCRs and, thus, that genome architectural features such as LCRs may have been instrumental in the generation of these CCRs.

Recurrent SOX9 deletion campomelic dysplasia due to somatic mosaicism in the father.

Haploinsufficiency of SOX9, a master gene in chondrogenesis and testis development, leads to the semi-lethal skeletal malformation syndrome campomelic dysplasia (CD), with or without XY sex reversal. We report on two children with CD and a phenotypically normal father, a carrier of a somatic mosaic SOX9 deletion. This is the first report of a mosaic deletion of SOX9; few familial CD cases with germline and somatic mutation mosaicism have been described. Our findings confirm the utility of aCGH and indicate that for a more accurate estimate of the recurrence risk for a completely penetrant autosomal dominant disorder, parental somatic mosaicism should be considered in healthy parents.

Two novel translocation breakpoints upstream of SOX9 define borders of the proximal and distal breakpoint cluster region in campomelic dysplasia.

The semilethal skeletal malformation syndrome campomelic dysplasia (CD) with or without XY sex reversal is caused by mutations within the SOX9 gene on 17q24.3 or by chromosomal aberrations (translocations, inversions or deletions) with breakpoints outside the SOX9 coding region. The previously published CD translocation breakpoints upstream of SOX9 fall into two clusters: a proximal cluster with breakpoints between 50-300 kb and a distal cluster with breakpoints between 899-932 kb. Here, we present clinical, cytogenetic and molecular data from two novel CD translocation cases. Case 1 with karyotype 46,XY,t(1;17)(q42.1;q24.3) has characteristic symptoms of CD, including mild tibial bowing, cryptorchidism and hypospadias. By standard fluorescence in situ hybridization (FISH) and by high-resolution fiber FISH, the 17q breakpoint was mapped 375 kb from SOX9, defining the centromeric border of the proximal breakpoint cluster region. Case 2 with karyotype 46,X,t(Y;17)(q11.2;q24.3) has the acampomelic form of CD and complete XY sex reversal. By FISH and somatic cell hybrid analysis, the 17q breakpoint was mapped 789 kb from SOX9, defining the telomeric border of the distal breakpoint cluster region. We discuss the structure of the 1 Mb cis-control region upstream of SOX9 and the correlation between the position of the 14 mapped translocation breakpoints with respect to disease severity and XY sex reversal.

Congenital anomalies and developmental delay in a boy with double chromosome 6 derived supernumerary marker.

The frequency of small supernumerary marker chromosomes has been estimated to approximately 0.45 per 1000 newborns. They are usually seen as single marker chromosomes in a mosaic state. Two cytogenetically identical markers have been observed only occasionally. We report on a boy, with congenital heart defect, neonatal hypotonia, hypogenitalism, delayed psychomotor development and mild dysmorphic facial features. The GTG karyotype performed on peripheral blood lymphocytes revealed a mosaic male karyotype with three cell lines. One cell line had a normal karyotype. In the other two either single or double chromosome 6 derived supernumerary markers were present, leading to partial trisomy or partial tetrasomy of chromosome 6, respectively.

The genomic basis of disease, mechanisms and assays for genomic disorders.

In the past fifteen years, an emerging group of genetic diseases have been described that result from DNA rearrangements rather than from single nucleotide changes. Such conditions have been referred to as genomic disorders. The predominant molecular mechanism underlying the rearrangements that cause this group of diseases and traits is nonallelic homologous recombination (NAHR) (unequal crossing-over between chromatids or chromosomes) utilizing low-copy repeats (LCRs) (also known as segmental duplications) as substrates. In contradistinction to highly repetitive sequences (e.g. Alu and LINE elements), these higher-order genomic architectural features usually span >1kb and up to hundreds of kilobases of genomic DNA, share >96% sequence identity and constitute >5% of the human genome. Many LCRs have complex structure and have arisen during primate speciation as a result of serial segmental duplications. LCRs can stimulate and/or mediate constitutional (both recurrent and nonrecurrent), evolutionary, and somatic rearrangements. Recently, copy-number variations (CNVs), also referred to as large-scale copy-number variations (LCVs) or copy-number polymorphisms (CNPs), parenthetically often associated with LCRs, have been demonstrated as a source of human variation as well as a potential cause of diseases. In addition to fluorescence in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE), and in silico analyses, multiplex ligation-dependent probe amplification (MLPA), and array comparative genomic hybridization (aCGH) with BAC and PAC clones have proven to be useful diagnostic methods for the detection and characterization of DNA rearrangements with the latter enabling high-resolution genome-wide analysis. The clinical implementation of such techniques is revolutionizing clinical cytogenetics.

A natural history of a child with monosomy 5p syndrome (Cat-cry/Cri-du-chat syndrome) during the 18 years of follow-up.

A record of a natural history of a long-term case study devoted to monosomy 5p (Cat-cry/Cri-du-chat) syndrome has been described rarely. Knowledge on the range of the changes in phenotype attributable to advancing age can be useful in clinical diagnosis of monosomy 5p at the different developmental stages, including adolescence, as well in prognosis for genetic counseling. In this case a detailed analysis of the morphologic phenotype in a girl with del(5)(p13.3) observed from 4 months to 18 years of age is reported. The comparative analysis of the girl's phenotype in different developmental stages has revealed that microcephaly, flat occipital region, face asymmetry, wide spaced palpebral fissures, epicanthic folds, small mouth fissure, thin mucous lip, small and low set ears and short IV metacarpals has not changed with advancing age. However, facial asymmetry was more evident, frontal tubers were less prominent, nasal root and back became prominent nasal back became elongated, the subnasal region was shorter and marked malocclusion appeared.

Congenital diaphragmatic hernia in WAGR syndrome.

Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome is a contiguous gene deletion syndrome involving the Wilms tumor 1 gene (WT1), the paired box gene 6 (PAX6), and possibly other genes on chromosome 11p13. WT1 is required for normal formation of the genitourinary system and the high incidence of Wilms tumor and genitourinary anomalies found in patients with WAGR are attributed to haploinsufficiency of this gene. It has been hypothesized that WT1 also plays an important role in the development of the diaphragm. During mammalian embryonic development, WT1 is expressed in the pleural and abdominal mesothelium that forms part of the diaphragm. Furthermore, mice that are homozygous for a deletion in the mouse homolog of WT1 have diaphragmatic hernias. Case reports describing congenital diaphragmatic hernias in infants with Denys-Drash and Frasier syndromes, both of which can be caused by mutations in WT1, provide additional support for this hypothesis. We report an infant with aniridia, bilateral cryptorchidism, vesicoureteral reflux, and a right-sided Morgagni-type diaphragmatic hernia. G-banded chromosome analysis revealed a deletion of 11p12-p15.1. Breakpoint regions were refined by fluorescence in situ hybridization (FISH) and deletion of the WAGR critical region, including WT1, was confirmed. A review of the medical literature identified a second patient with a deletion of 11p13, a left-sided Bochdalek-type diaphragmatic hernia, and anomalies that suggest a diagnosis of WAGR including bilateral microphthalmia, a small penis, bilateral cryptorchidism, and a hypoplastic scrotum. These cases demonstrate that congenital diaphragmatic hernia can be associated with WAGR syndrome and suggest that deletions of WT1 may predispose individuals to develop congenital diaphragmatic hernia.

Interstitial deletion of 10p and atrial septal defect in DiGeorge 2 syndrome.

We present molecular genetic investigations of a 4-year-old boy with craniofacial dysmorphism and developmental delay. Trivial mitral and tricuspid regurgitation without gross structural abnormality was diagnosed by echocardiography. High-resolution chromosome analysis revealed an interstitial deletion, del(10)(p12.1p12.32). To characterize the deletion size and breakpoints, we performed fluorescence in situ hybridization analysis using 27 BAC clones. Our data demonstrate an approximately 5.5 Mb deletion del(10)(p12.1p12.31). Surprisingly, the BAC clone RP11-56H7 that contains NEBL, an apparent downstream gene of the cardiogenic transcription factor HAND2 previously shown to be deleted in the patients with DiGeorge 2 syndrome and 10p13 deletion, was deleted in our patient with 10p12.1-p12.31 deletion. In addition, we provide clinical data and results of molecular analysis for a patient with multiple congenital anomalies including Ebstein's anomaly, kidney malformations, and 10p13-p14 deletion. We also reviewed 19 patients with congenital heart defects and deletions involving 10p and propose that atrial septal defect (ASD) is a common cardiac anomaly associated with DiGeorge 2 syndrome. Based on genotype-phenotype analysis of published patients and those reported herein, we propose an approximately 1.0 Mb critical region between loci D10S547 and D10S2176 in 10p14 to be associated with ASD. Considering that septal defects are the most frequent congenital heart anomaly, we suggest that further investigations in the 10p critical region are important to identify gene(s) responsible for this common birth defect.