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The developmental disorder Burn-McKeown Syndrome (BMKS) is characterised by choanal atresia and specific craniofacial features. BMKS is caused by biallelic variants in the pre-messenger RNA splicing factor TXNL4A. Most patients have a loss-of-function variant in trans with a 34-base pair (bp) deletion (type 1 Δ34) in the promoter region. Here, we identified two patients with BMKS. One individual has a TXNL4A c.93_94delCC, p.His32Argfs*21 variant combined with a type 1 Δ34 promoter deletion. The other has an intronic TXNL4A splice site variant (c.258-3C>G) and a type 1 Δ34 promoter deletion. We show the c.258-3C>G variant, and a previously reported c.258-2A>G variant, cause skipping of the final exon of TXNL4A in a minigene splicing assay. Furthermore, we identify putative transcription factor binding sites within the 56bp of the TXNL4A promoter affected by the type 1 and type 2 Δ34 and use dual luciferase assays to identify a 22bp repeated motif essential for TXNL4A expression within this promoter region. We propose that additional variants affecting critical transcription factor binding nucleotides within the 22bp repeated motif could be relevant to BMKS etiology. Finally, our data emphasizes the need to analyse the non-coding sequence in individuals where a single likely pathogenic coding variant is identified in an autosomal recessive disorder consistent with the clinical presentation.

De novo pathogenic variants in the additional sex combs-like 3 (ASXL3) gene cause a rare multi-systemic neurodevelopmental disorder. There is growing evidence that germline and somatic mosaicism are more common and play a greater role in genetic disorders than previously acknowledged. There is one previous report of ASXL3-related syndrome caused by de novo pathogenic variants in two siblings suggesting gonadal mosaicism. In this report, we present five patients with ASXL3-related syndrome, describing two families comprising two non-twin siblings harbouring apparent de novo pathogenic variants in ASXL3. Parents were clinically unaffected and there was no evidence of mosaicism from genomic DNA on exome-trio data, suggesting germline mosaicism in one of the parents. We also describe clinical details of a patient with typical features of ASXL3-related syndrome and mosaic de novo pathogenic variant in ASXL3 in 30-35% of both blood and saliva sample on trio-exome sequencing. We expand the known genetic basis of ASXL3-related syndromes and discuss mosaicism as a disease mechanism in five patients from three unrelated families. The findings of this report highlight the importance of taking gonadal mosaicism into consideration when counselling families regarding recurrence risk. We also discuss postzygotic mosaicism as a cause of fully penetrant ASXL3-related syndrome.

PURPOSE: Pathogenic variants in the chromatin organizer CTCF were previously reported in seven individuals with a neurodevelopmental disorder (NDD). METHODS: Through international collaboration we collected data from 39 subjects with variants in CTCF. We performed transcriptome analysis on RNA from blood samples and utilized Drosophila melanogaster to investigate the impact of Ctcf dosage alteration on nervous system development and function. RESULTS: The individuals in our cohort carried 2 deletions, 8 likely gene-disruptive, 2 splice-site, and 20 different missense variants, most of them de novo. Two cases were familial. The associated phenotype was of variable severity extending from mild developmental delay or normal IQ to severe intellectual disability. Feeding difficulties and behavioral abnormalities were common, and variable other findings including growth restriction and cardiac defects were observed. RNA-sequencing in five individuals identified 3828 deregulated genes enriched for known NDD genes and biological processes such as transcriptional regulation. Ctcf dosage alteration in Drosophila resulted in impaired gross neurological functioning and learning and memory deficits. CONCLUSION: We significantly broaden the mutational and clinical spectrum ofCTCF-associated NDDs. Our data shed light onto the functional role of CTCF by identifying deregulated genes and show that Ctcf alterations result in nervous system defects in Drosophila. ispartof: GENETICS IN MEDICINE vol:21 issue:12 pages:2723-2733 ispartof: location:United States status: published

Mobile genetic Elements (MEs) are segments of DNA which can copy themselves and other transcribed sequences through the process of retrotransposition (RT). In humans several disorders have been attributed to RT, but the role of RT in severe developmental disorders (DD) has not yet been explored. Here we identify RT-derived events in 9738 exome sequenced trios with DD-affected probands. We ascertain 9 de novo MEs, 4 of which are likely causative of the patient’s symptoms (0.04%), as well as 2 de novo gene retroduplications. Beyond identifying likely diagnostic RT events, we estimate genome-wide germline ME mutation rate and selective constraint and demonstrate that coding RT events have signatures of purifying selection equivalent to those of truncating mutations. Overall, our analysis represents a comprehensive interrogation of the impact of retrotransposition on protein coding genes and a framework for future evolutionary and disease studies., Retrotransposition events have been linked to some human disorders. Here, Gardner et al. systematically search for mobile genetic elements (ME) in trio whole exome-sequencing datasets and ascertain 9 de novo MEs and further estimate genome-wide germline ME burden and constraint.

Alexander disease (AD) is a leukodystrophy caused by heterozygous mutations in the gene encoding the glial fibrillary acidic protein (GFAP). Currently, de novo heterozygous missense mutations in the GFAP gene are identified in over 95% of patients with AD. However, patients with biopsy-proven AD have been reported in whom no GFAP mutation has been identified. We report identical twin boys presenting in infancy with seizures and developmental delay in whom MR appearances were suggestive of AD with the exception of an unusual, bilateral, arc of calcification at the frontal white–gray junction. Initial mutation screening of the GFAP gene did not identify a mutation. Whole exome sequencing in both brothers revealed a de novo heterozygous in-frame deletion of the whole of exon 5 of the GFAP gene. Mutations in the GFAP gene are thought to result in a toxic effect of mutant GFAP disrupting the formation of the normal intermediate filament network and resulting in Rosenthal fiber formation, which has hitherto not been linked to exonic scale copy number variants in GFAP. Further studies on mutation negative AD patients are warranted to determine whether a similar mechanism underlies their disease.

PurposeWe delineate the clinical spectrum and describe the histology in arterial tortuosity syndrome (ATS), a rare connective tissue disorder characterized by tortuosity of the large and medium-sized arteries, caused by mutations in SLC2A10.MethodsWe retrospectively characterized 40 novel ATS families (50 patients) and reviewed the 52 previously reported patients. We performed histology and electron microscopy (EM) on skin and vascular biopsies and evaluated TGF-β signaling with immunohistochemistry for pSMAD2 and CTGF.ResultsStenoses, tortuosity, and aneurysm formation are widespread occurrences. Severe but rare vascular complications include early and aggressive aortic root aneurysms, neonatal intracranial bleeding, ischemic stroke, and gastric perforation. Thus far, no reports unequivocally document vascular dissections or ruptures. Of note, diaphragmatic hernia and infant respiratory distress syndrome (IRDS) are frequently observed. Skin and vascular biopsies show fragmented elastic fibers (EF) and increased collagen deposition. EM of skin EF shows a fragmented elastin core and a peripheral mantle of microfibrils of random directionality. Skin and end-stage diseased vascular tissue do not indicate increased TGF-β signaling.ConclusionOur findings warrant attention for IRDS and diaphragmatic hernia, close monitoring of the aortic root early in life, and extensive vascular imaging afterwards. EM on skin biopsies shows disease-specific abnormalities.GENETICS in MEDICINE advance online publication, 11 January 2018; doi:10.1038/gim.2017.253. ispartof: Genetics in Medicine vol:20 issue:10 pages:1236-1245 ispartof: location:United States status: published

Histone lysine methyltransferases (KMTs) and demethylases (KDMs) underpin gene regulation. Here we demonstrate that variants causing haploinsufficiency of KMTs and KDMs are frequently encountered in individuals with developmental disorders. Using a combination of human variation databases and existing animal models, we determine 22 KMTs and KDMs as additional candidates for dominantly inherited developmental disorders. We show that KMTs and KDMs that are associated with, or are candidates for, dominant developmental disorders tend to have a higher level of transcription, longer canonical transcripts, more interactors, and a higher number and more types of post-translational modifications than other KMT and KDMs. We provide evidence to firmly associate KMT2C, ASH1L, and KMT5B haploinsufficiency with dominant developmental disorders. Whereas KMT2C or ASH1L haploinsufficiency results in a predominantly neurodevelopmental phenotype with occasional physical anomalies, KMT5B mutations cause an overgrowth syndrome with intellectual disability. We further expand the phenotypic spectrum of KMT2B-related disorders and show that some individuals can have severe developmental delay without dystonia at least until mid-childhood. Additionally, we describe a recessive histone lysine-methylation defect caused by homozygous or compound heterozygous KDM5B variants and resulting in a recognizable syndrome with developmental delay, facial dysmorphism, and camptodactyly. Collectively, these results emphasize the significance of histone lysine methylation in normal human development and the importance of this process in human developmental disorders. Our results demonstrate that systematic clinically oriented pathway-based analysis of genomic data can accelerate the discovery of rare genetic disorders.

Contains fulltext : 152973.pdf (Publisher’s version ) (Closed access) Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.

Autosomal recessive cutis laxa type I (ARCL type I) is characterized by generalized cutis laxa with pulmonary emphysema and/or vascular complications. Rarely, mutations can be identified in FBLN4 or FBLN5. Recently, LTBP4 mutations have been implicated in a similar phenotype. Studying FBLN4, FBLN5, and LTBP4 in 12 families with ARCL type I, we found bi-allelic FBLN5 mutations in two probands, whereas nine probands harbored biallelic mutations in LTBP4. FBLN5 and LTBP4 mutations cause a very similar phenotype associated with severe pulmonary emphysema, in the absence of vascular tortuosity or aneurysms. Gastrointestinal and genitourinary tract involvement seems to be more severe in patients with LTBP4 mutations. Functional studies showed that most premature termination mutations in LTBP4 result in severely reduced mRNA and protein levels. This correlated with increased transforming growth factor-beta (TGFβ) activity. However, one mutation, c.4127dupC, escaped nonsense-mediated decay. The corresponding mutant protein (p.Arg1377Alafs(*) 27) showed reduced colocalization with fibronectin, leading to an abnormal morphology of microfibrils in fibroblast cultures, while retaining normal TGFβ activity. We conclude that LTBP4 mutations cause disease through both loss of function and gain of function mechanisms.

Audo, Isabelle, et al., Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated., The project was supported by GIS-maladies rares (C.Z.), Retina France ([part of the 100-Exome Project] I.A., C.P.H., J.-A.S., H.D. and C.Z.), Foundation Voir et Entendre (C.Z.), Agence National de la Recherche (S.S.B), Foundation Fighting Blindness (FFB) grant CD-CL-0808-0466-CHNO (I.A. and the CIC503, recognized as an FFB center), FFB grant C-CMM-0907-0428-INSERM04, Ville de Paris and Région Ille de France, the French Association against Myopathy (AFM) grant KBM-14390 (O.P.), and National Institutes of Health grant 1R01EY020902-01A1 (K.B.).

The NONO protein has been characterized as an important transcriptional regulator in diverse cellular contexts. Here we show that loss of NONO function is a likely cause of human intellectual disability and that NONO-deficient mice have cognitive and affective deficits. Correspondingly, we find specific defects at inhibitory synapses, where NONO regulates synaptic transcription and gephyrin scaffold structure. Our data identify NONO as a possible neurodevelopmental disease gene and highlight the key role of the DBHS protein family in functional organization of GABAergic synapses.

The X-linked dominant trait focal dermal hypoplasia (FDH, Goltz syndrome) is a developmental defect with focal distribution of affected tissues due to a block of Wnt signal transmission from cells carrying a detrimental PORCN mutation on an active X-chromosome. Molecular characterization of 24 unrelated patients from different ethnic backgrounds revealed 23 different mutations of the PORCN gene in Xp11.23. Three were microdeletions eliminating PORCN and encompassing neighboring genes such as EBP, the gene associated with Conradi-Hunermann-Happle syndrome (CDPX2). 12/24 patients carried nonsense mutations resulting in loss of function. In one case a canonical splice acceptor site was mutated, and 8 missense mutations exchanged highly conserved amino acids. FDH patients overcome the consequences of potentially lethal X-chromosomal mutations by extreme skewing of X-chromosome inactivation in females, enabling transmission of the trait in families, or by postzygotic mosaicism both in male and female individuals. Molecular characterization of the PORCN mutations in cases diagnosed as Goltz syndrome is particularly relevant for genetic counseling of patients and their families since no functional diagnostic test is available and carriers of the mutation might otherwise be overlooked due to considerable phenotypic variability associated with the mosaic status.

Congenital Heart Disease (CHD) is the most common congenital defect. Many families with left-right laterality defects and complex CHD have an unknown genetic aetiology. Many ciliopathies, including Primary Ciliary Dyskinesia (PCD), are associated with intracardiac defects. The role of primary cilia in cardiac morphogenesis remains unknown, although cardiac cilia have roles that are distinct from the definition of laterality at the embryonic node. We hypothesise that defects in genes important in the assembly and function of cilia are responsible for some inherited forms of CHD. This research project aims to recruit families with a recurrence of CHD and to perform Whole Exome Sequencing (WES) to identify putative pathogenic variants and to delineate novel genetic causes of CHD. Twelve families have now been recruited and WES has been carried out in seven of these families using paired-end sequencing. Data analysis follows a standardised pipeline to call and then filter variants in order to assess their pathogenic potential. Variants are prioritized on the basis of known or suspected function of the encoded protein, and publicly-available RNA expression data. Variant filtering has allowed the identification of a limited number of candidate variants in recruited families. Of particular interest, a likely causative homozygous variant within a PCD gene has been identified in two siblings affected with heterotaxy, thus confirming a link between ciliopathies and CHD. The function and interactions of identified genes will be assessed, using cellular techniques and animal models, to provide insights into the pathogenesis of CHD.

Purpose. To identify the defective gene in the sex-linked, recessively inherited retinal dysplasia and degeneration (rdd) chicken and to search for the human equivalent disease. Methods. Microsatellites from chicken chromosome Z were genotyped in 77 progeny of a carrier male (rdd/+) and an affected female (rdd/W), and candidate genes were sequenced. Retinal cross-sections from rdd and wild-type birds were analyzed by immunohistology. The human orthologous gene was screened in a panel of archival DNAs from 276 patients with retinitis pigmentosa (RP) or Leber congenital amaurosis (LCA) using melting curve analysis and DNA sequencing. Results. The rdd locus was refined to an approximately 3-Mb region on chromosome Z. Sequence analysis identified a C→T change in the mpdz gene that created a premature stop codon (c.1372C→T, p.R458X), which segregated with the disease phenotype. As expected, the full-length mpdz protein was absent in rdd retinas, but in wild-type birds, it localized to the retinal outer limiting membrane, where it may have a role in the interactions between photoreceptors and Muller glia cells. The screen to identify the human equivalent disease found 10 heterozygous variants in the orthologous gene in patients with RP (three missense and two null alleles) and LCA (four missense and one null allele). Conclusions. These findings reveal that MPDZ is essential for normal development of the retina and may have a role in maintaining photoreceptor integrity. The identification of human mutations suggests that MPDZ plays a role in human retinal disease, but the precise nature of this role remains to be determined.

BACKGROUND: Constitutional DICER1 mutations were recently reported to cause familial pleuropulmonary blastoma (PPB). AIM: To investigate the contribution and phenotypic spectrum of constitutional and somatic DICER1 mutations to cancer. METHODS AND RESULTS: The authors sequenced DICER1 in constitutional DNA from 823 unrelated patients with a variety of tumours and in 781 cancer cell lines. Constitutional DICER1 mutations were identified in 19 families including 11/14 with PPB, 2/3 with cystic nephroma, 4/7 with ovarian Sertoli-Leydig-type tumours, 1/243 with Wilms tumour (this patient also had a Sertoli-Leydig tumour), 1/1 with intraocular medulloepithelioma (this patient also had PPB), 1/86 with medulloblastoma/infratentorial primitive neuroectodermal tumour, and 1/172 with germ cell tumour. The inheritance was investigated in 17 families. DICER1 mutations were identified in 25 relatives: 17 were unaffected, one mother had ovarian Sertoli-Leydig tumour, one half-sibling had cystic nephroma, and six relatives had non-toxic thyroid cysts/goitre. Analysis of eight tumours from DICER1 mutation-positive patients showed universal retention of the wild-type allele. DICER1 truncating mutations were identified in 4/781 cancer cell lines; all were in microsatellite unstable lines and therefore unlikely to be driver mutations. CONCLUSION: Constitutional DICER1 haploinsufficiency predisposes to a broad range of tumours, making a substantial contribution to PPB, cystic nephroma and ovarian Sertoli-Leydig tumours, but a smaller contribution to other tumours. Most mutation carriers are unaffected, indicating that tumour risk is modest. The authors define the clinical contexts in which DICER1 mutation testing should be considered, the associated tumour risks, and the implications for at-risk individuals. They have termed this condition 'DICER1 syndrome'. ACCESSION NUMBERS: The cDNA Genbank accession number for the DICER1 sequence reported in this paper is NM_030621.2.

Ulnar Mammary syndrome (UMS) is an autosomal disorder caused by haploinsufficiency of the TBX3 gene. There is marked intrafamilial variation in expression of the syndrome. We present one three generation family in which the proband has absence of the right ulna and third, fourth and fifth rays in her right hand. Her mother and maternal grandmother have more subtle anomalies while all have a similar facial appearance with a broad nasal tip, a broad jaw, a prominent chin and a tongue frenulum. They have a single base pair insertion (c. 992dup) in TBX3. We compare faces from the handful of published UMS patients which include photographs, this family and four other cases with TBX3 mutations. All have similarities in appearance which we suggest could alert clinicians to the possibility of a TBX3 mutation if individuals present with more subtle features of UMS such as postaxial polydactyly, isolated 5th finger anomalies, delayed puberty in males, breast hypoplasia or short stature with or without growth hormone deficiency.

Aortic arch artery patterning defects account for approximately 20% of congenital cardiovascular malformations and are observed frequently in velocardiofacial syndrome (VCFS). In the current study, we screened for chromosome rearrangements in patients suspected of VCFS, but who lacked a 22q11 deletion or TBX1 mutation. One individual displayed hemizygous CHD7, which encodes a chromodomain protein. CHD7 haploinsufficiency is the major cause of coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, and ear anomalies/deafness (CHARGE) syndrome, but this patient lacked the major diagnostic features of coloboma and choanal atresia. Because a subset of CHARGE cases also display 22q11 deletions, we explored the embryological relationship between CHARGE and VCSF using mouse models. The hallmark of Tbx1 haploinsufficiency is hypo/aplasia of the fourth pharyngeal arch artery (PAA) at E10.5. Identical malformations were observed in Chd7 heterozygotes, with resulting aortic arch interruption at later stages. Other than Tbx1, Chd7 is the only gene reported to affect fourth PAA development by haploinsufficiency. Moreover, Tbx1+/–;Chd7+/– double heterozygotes demonstrated a synergistic interaction during fourth PAA, thymus, and ear morphogenesis. We could not rescue PAA morphogenesis by restoring neural crest Chd7 expression. Rather, biallelic expression of Chd7 and Tbx1 in the pharyngeal ectoderm was required for normal PAA development.

Mutations in the transcription factor encoding TFAP2A gene underlie branchio-oculo-facial syndrome (BOFS), a rare dominant disorder characterized by distinctive craniofacial, ocular, ectodermal and renal anomalies. To elucidate the range of ocular phenotypes caused by mutations in TFAP2A, we took three approaches. First, we screened a cohort of 37 highly selected individuals with severe ocular anomalies plus variable defects associated with BOFS for mutations or deletions in TFAP2A. We identified one individual with a de novo TFAP2A four amino acid deletion, a second individual with two non-synonymous variations in an alternative splice isoform TFAP2A2, and a sibling-pair with a paternally inherited whole gene deletion with variable phenotypic expression. Second, we determined that TFAP2A is expressed in the lens, neural retina, nasal process, and epithelial lining of the oral cavity and palatal shelves of human and mouse embryos--sites consistent with the phenotype observed in patients with BOFS. Third, we used zebrafish to examine how partial abrogation of the fish ortholog of TFAP2A affects the penetrance and expressivity of ocular phenotypes due to mutations in genes encoding bmp4 or tcf7l1a. In both cases, we observed synthetic, enhanced ocular phenotypes including coloboma and anophthalmia when tfap2a is knocked down in embryos with bmp4 or tcf7l1a mutations. These results reveal that mutations in TFAP2A are associated with a wide range of eye phenotypes and that hypomorphic tfap2a mutations can increase the risk of developmental defects arising from mutations at other loci.

The occurrence of familial 22q11 deletion syndrome (22q11DS) raises the possibility of prenatal diagnosis for those families at risk, and parents of any new case should be offered deletion screening. Deletion of 22q11 is the most frequent interstitial chromosome deletion observed in man, begging the question as to whether there is any structural predisposition to chromosome rearrangements of this region. The single gene hypothesis predicted that a subset of those velo-cardio-facial syndrome/DiGeorge Syndrome (VCFS/DGS) patients with no apparent deletion of 22q11 would have a small deletion or point mutation inactivating the major gene haploinsufficient in the condition. The frustration at failure to find any loss of function mutations of DGCR genes in patients with no deletion prompted investigators to pursue animal models. Given the cognitive deficits and increased incidence of behavioral difficulty in 22q11DS attempts have been made to identify behavioral correlates in the mouse deletion model.

Many genetic syndromes involve a facial gestalt that suggests a preliminary diagnosis to an experienced clinical geneticist even before a clinical examination and genotyping are undertaken. Previously, using visualization and pattern recognition, we showed that dense surface models (DSMs) of full face shape characterize facial dysmorphology in Noonan and in 22q11 deletion syndromes. In this much larger study of 696 individuals, we extend the use of DSMs of the full face to establish accurate discrimination between controls and individuals with Williams, Smith-Magenis, 22q11 deletion, or Noonan syndromes and between individuals with different syndromes in these groups. However, the full power of the DSM approach is demonstrated by the comparable discriminating abilities of localized facial features, such as periorbital, perinasal, and perioral patches, and the correlation of DSM-based predictions and molecular findings. This study demonstrates the potential of face shape models to assist clinical training through visualization, to support clinical diagnosis of affected individuals through pattern recognition, and to enable the objective comparison of individuals sharing other phenotypic or genotypic properties.

The 22q11 deletion syndrome (22q11DS; DiGeorge/velo-cardio-facial syndrome) primarily affects the structures comprising the pharyngeal arches and pouches resulting in arch artery, cardiac, parathyroid, thymus, palatal and craniofacial defects. Tbx1 haploinsufficiency is thought to account for the main structural anomalies observed in the 22q11DS. The Df1 deleted mouse provides a model for 22q11DS, the deletion reflecting Tbx1 haploinsufficiency in the context of the deletion of 21 adjacent genes. We examined the expression of genes in Df1 embryos at embryonic day (E) 10.5, a stage when the arch-artery phenotype is fully penetrant. Our aims were threefold, with our primary aim to identify differentially regulated genes. Second, we asked whether any of the genes hemizygous in Df1 were dosage compensated to wild type levels, and third we investigated whether genes immediately adjacent to the deletion were dysregulated secondary to a position effect. Utilisation of oligonulceotide arrays allowed us to achieve our aims with 9 out of 12 Df1 deleted genes passing the stringent statistical filtering applied. Several genes involved in vasculogenesis and cardiogenesis were validated by real time quantitative PCR (RTQPCR), including Connexin 45, a gene required for normal vascular development, and Dnajb9 a gene implicated in microvascular differentiation. There was no evidence of any dosage compensation of deleted genes, suggesting this phenomenon is rare, and no dysregulation of genes mapping immediately adjacent to the deletion was detected. However Crkl, another gene implicated in the 22q11DS phenotype, was found to be downregulated by microarray and RTQPCR.

The 22q11 deletion syndrome (22q11DS) is a developmental syndrome comprising of heart, palate, thymus and parathyroid glands defects. Individuals with 22q11DS usually carry a 1.5- to 3-Mb heterozygous deletion on chromosome 22q11.2. However, there are many patients with features of 22q11DS without a known cause from conventional karyotype and FISH analysis. Six patients with features of 22q11DS, a normal chromosomal and FISH 22q11 analysis, were selected for investigation by microarray genomic comparative hybridisation (array CGH). Array-CGH is a powerful technology enabling detection of submicroscopic chromosome duplications and deletions by comparing a differentially labelled test sample to a control. The samples are co-hybridised to a microarray containing genomic clones and the resulting ratio of fluorescence intensities on each array element is proportional to the DNA copy number difference. No chromosomal changes were detected by hybridisation to a high resolution array representing chromosome 22q. However, one patient was found to have a 6-Mb deletion on 5q11.2 detected by a whole genome 1-Mb array. This deletion was confirmed with fluorescence in-situ hybridisation (FISH) and microsatellite marker analysis. It is the first deletion described in this region. The patient had tetralogy of Fallot, a bifid uvula and velopharyngeal insufficiency, short stature, learning and behavioural difficulties. This case shows the increased sensitivity of array CGH over detailed karyotype analysis for detection of chromosomal changes. It is anticipated that array CGH will improve the clinician's capacity to diagnose congenital syndromes with an unknown aetiology.

A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR–Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR–ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR–ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP–deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR–deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR–ATRIP deficient sub-class of Seckel Syndrome. ATR–ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR–SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR–ATRIP Seckel Syndrome to be defined., Author Summary Seckel Syndrome (SS) is a rare human disorder characterised by small head circumference and delayed growth. Patients can show additional features including abnormal bone development, receding chins, sloping foreheads, and small ears. In 2003, we identified ataxia telangiectasia and Rad3 related (ATR) as a causal genetic defect in two related families displaying SS. However, additional patients with mutations in ATR have not hitherto been identified. Here, we describe two further patients with novel mutations in ATR. Additionally, we identify a patient with mutations in ATRIP, which encodes an interacting partner of ATR, representing a novel genetic defect causing SS. ATR functions to promote the ability of cells to recover from difficulties encountered during replication. We show that patient-derived cells have reduced ATR and ATRIP protein levels and defective ATR/ATRIP function. Our identification of further ATR–ATRIP defective patients and a consideration of their clinical features aids the characterisation and identification of this form of SS and provides insight into the role played by the ATR–ATRIP complex during development.

Individuals with severe, undiagnosed developmental disorders (DDs) are enriched for damaging de novo mutations (DNMs) in developmentally important genes. We exome sequenced 4,293 families with individuals with DDs, and meta-analysed these data with published data on 3,287 individuals with similar disorders. We show that the most significant factors influencing the diagnostic yield of de novo mutations are the sex of the affected individual, the relatedness of their parents and the age of both father and mother. We identified 94 genes enriched for damaging de novo mutation at genome-wide significance (P < 7 × 10−7), including 14 genes for which compelling data for causation was previously lacking. We have characterised the phenotypic diversity among these genetic disorders. We demonstrate that, at current cost differentials, exome sequencing has much greater power than genome sequencing for novel gene discovery in genetically heterogeneous disorders. We estimate that 42% of our cohort carry pathogenic DNMs (single nucleotide variants and indels) in coding sequences, with approximately half operating by a loss-of-function mechanism, and the remainder resulting in altered-function (e.g. activating, dominant negative). We established that most haplo insufficient developmental disorders have already been identified, but that many altered-function disorders remain to be discovered. Extrapolating from the DDD cohort to the general population, we estimate that developmental disorders caused by DNMs have an average birth prevalence of 1 in 213 to 1 in 448 (0.22-0.47% of live births), depending on parental age.AbbreviationsPTVProtein-Truncating VariantDNMDe Novo MutationDDDevelopmental DisorderDDDDeciphering Developmental Disorders study