Search

An increasing number of individuals with intellectual developmental disorder (IDD) and heterozygous variants in BCL11A are identified, yet our knowledge of manifestations and mutational spectrum is lacking. To address this, we performed detailed analysis of 42 individuals with BCL11A-related IDD (BCL11A-IDD, a.k.a. Dias-Logan syndrome) ascertained through an international collaborative network, and reviewed 35 additional previously reported patients. Analysis of 77 affected individuals identified 60 unique disease-causing variants (30 frameshift, 7 missense, 6 splice-site, 17 stop-gain) and 8 unique BCL11A microdeletions. We define the most prevalent features of BCL11A-IDD: IDD, postnatal-onset microcephaly, hypotonia, behavioral abnormalities, autism spectrum disorder, and persistence of fetal hemoglobin (HbF), and identify autonomic dysregulation as new feature. BCL11A-IDD is distinguished from 2p16 microdeletion syndrome, which has a higher incidence of congenital anomalies. Our results underscore BCL11A as an important transcription factor in human hindbrain development, identifying a previously underrecognized phenotype of a small brainstem with a reduced pons/medulla ratio. Genotype-phenotype correlation revealed an isoform-dependent trend in severity of truncating variants: those affecting all isoforms are associated with higher frequency of hypotonia, and those affecting the long (BCL11A-L) and extra-long (-XL) isoforms, sparing the short (-S), are associated with higher frequency of postnatal microcephaly. With the largest international cohort to date, this study highlights persistence of fetal hemoglobin as a consistent biomarker and hindbrain abnormalities as a common feature. It contributes significantly to our understanding of BCL11A-IDD through an extensive unbiased multi-center assessment, providing valuable insights for diagnosis, management and counselling, and into BCL11A's role in brain development., (© 2024. The Author(s).)

Pathogenic SOX11 variants have been associated with intellectual developmental disorder with microcephaly, and with or without ocular malformations or hypogonadotropic hypogonadism (HH) (IDDMOH, OMIM # 615866). In this article, we report seven new patients with de novo SOX11 variants. Five of the variants are missense, one nonsense, and one whole-gene deletion, most of them are novel variants. The main clinical features included neurodevelopmental delay (7/7) and intellectual disability (5/7), autism/attention deficit hyperactivity disorder (5/7), microcephaly (4/7), short stature (4/7), hypotonia (4/7), and clinodactyly of the 5th fingers (5/7). HH was confirmed in two female patients with primary amenorrhea, nonvisualized/prepubertal size of the uterus, and nonvisualized ovaries. Two of the male patients presented with micropenis, two had cryptorchidism, and one had decreased testicular size, which are suggestive findings of HH. This article contributes to the clinical characterization of patients with SOX11 variants and supports the role of this gene in HH., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

Mobile element insertions (MEIs) are a known cause of genetic disease but have been underexplored due to technical limitations of genetic testing methods. Various bioinformatic tools have been developed to identify MEIs in Next Generation Sequencing data. However, most tools have been developed specifically for genome sequencing (GS) data rather than exome sequencing (ES) data, which remains more widely used for routine diagnostic testing. In this study, we benchmarked six MEI detection tools (ERVcaller, MELT, Mobster, SCRAMble, TEMP2 and xTea) on ES data and on GS data from publicly available genomic samples (HG002, NA12878). For all the tools we evaluated sensitivity and precision of different filtering strategies. Results show that there were substantial differences in tool performance between ES and GS data. MELT performed best with ES data and its combination with SCRAMble increased substantially the detection rate of MEIs. By applying both tools to 10,890 ES samples from Solve-RD and 52,624 samples from Radboudumc we were able to diagnose 10 patients who had remained undiagnosed by conventional ES analysis until now. Our study shows that MELT and SCRAMble can be used reliably to identify clinically relevant MEIs in ES data. This may lead to an additional diagnosis for 1 in 3000 to 4000 patients in routine clinical ES., (© 2023. The Author(s).)

Various groups of neurological disorders, including movement disorders and neuromuscular diseases, are clinically and genetically heterogeneous. Diagnostic panel-based exome sequencing is a routine test for these disorders. Despite the success rates of exome sequencing, it results in the detection of causative sequence variants in 'only' 25-30% of cases. Copy number variants (CNVs), i.e. deletion or duplications, explain 10-20% of individuals with multisystemic phenotypes, such as co-existing intellectual disability, but may also have a role in disorders affecting a single system (organ), like neurological disorders with normal intelligence. In this study, CNVs were extracted from clinical exome sequencing reports of 4800 probands primarily with a movement disorder, myopathy or neuropathy. In 88 (~2%) probands, phenotype-matching CNVs were detected, representing ~7% of genetically confirmed cases. CNVs varied from involvement of over 100 genes to single exons and explained X-linked, autosomal dominant, or - recessive disorders, the latter due to either a homozygous CNV or a compound heterozygous CNV with a sequence variant on the other allele. CNVs were detected affecting genes where deletions or duplications are established as a common mechanism, like PRKN (in Parkinson's disease), DMD (in Duchenne muscular dystrophy) and PMP22 (in neuropathies), but also genes in which no intragenic CNVs have been reported to date. Analysis of CNVs as part of panel-based exome sequencing for genetically heterogeneous neurological diseases provides an additional diagnostic yield of ~2% without extra laboratory costs. Therefore it is recommended to perform CNV analysis for movement disorders, muscle disease, neuropathies, or any other single-system disorder., (© 2023. The Author(s).)

Genome sequencing (GS) can identify novel diagnoses for patients who remain undiagnosed after routine diagnostic procedures. We tested whether GS is a better first-tier genetic diagnostic test than current standard of care (SOC) by assessing the technical and clinical validity of GS for patients with neurodevelopmental disorders (NDD). We performed both GS and exome sequencing in 150 consecutive NDD patient-parent trios. The primary outcome was diagnostic yield, calculated from disease-causing variants affecting exonic sequence of known NDD genes. GS (30%, n = 45) and SOC (28.7%, n = 43) had similar diagnostic yield. All 43 conclusive diagnoses obtained with SOC testing were also identified by GS. SOC, however, required integration of multiple test results to obtain these diagnoses. GS yielded two more conclusive diagnoses, and four more possible diagnoses than ES-based SOC (35 vs. 31). Interestingly, these six variants detected only by GS were copy number variants (CNVs). Our data demonstrate the technical and clinical validity of GS to serve as routine first-tier genetic test for patients with NDD. Although the additional diagnostic yield from GS is limited, GS comprehensively identified all variants in a single experiment, suggesting that GS constitutes a more efficient genetic diagnostic workflow., (© 2022. The Author(s).)

Witteveen-Kolk syndrome (OMIM 613406) is a recently defined neurodevelopmental syndrome caused by heterozygous loss-of-function variants in SIN3A. We define the clinical and neurodevelopmental phenotypes related to SIN3A-haploinsufficiency in 28 unreported patients. Patients with SIN3A variants adversely affecting protein function have mild intellectual disability, growth and feeding difficulties. Involvement of a multidisciplinary team including a geneticist, paediatrician and neurologist should be considered in managing these patients. Patients described here were identified through a combination of clinical evaluation and gene matching strategies (GeneMatcher and Decipher). All patients consented to participate in this study. Mean age of this cohort was 8.2 years (17 males, 11 females). Out of 16 patients ≥ 8 years old assessed, eight (50%) had mild intellectual disability (ID), four had moderate ID (22%), and one had severe ID (6%). Four (25%) did not have any cognitive impairment. Other neurological symptoms such as seizures (4/28) and hypotonia (12/28) were common. Behaviour problems were reported in a minority. In patients ≥2 years, three were diagnosed with Autism Spectrum Disorder (ASD) and four with Attention Deficit Hyperactivity Disorder (ADHD). We report 27 novel variants and one previously reported variant. 24 were truncating variants; three were missense variants and one large in-frame gain including exons 10-12.

De novo mutations in protein-coding genes are a well-established cause of developmental disorders 1 . However, genes known to be associated with developmental disorders account for only a minority of the observed excess of such de novo mutations 1,2 . Here, to identify previously undescribed genes associated with developmental disorders, we integrate healthcare and research exome-sequence data from 31,058 parent-offspring trios of individuals with developmental disorders, and develop a simulation-based statistical test to identify gene-specific enrichment of de novo mutations. We identified 285 genes that were significantly associated with developmental disorders, including 28 that had not previously been robustly associated with developmental disorders. Although we detected more genes associated with developmental disorders, much of the excess of de novo mutations in protein-coding genes remains unaccounted for. Modelling suggests that more than 1,000 genes associated with developmental disorders have not yet been described, many of which are likely to be less penetrant than the currently known genes. Research access to clinical diagnostic datasets will be critical for completing the map of genes associated with developmental disorders.

Determining pathogenicity of genomic variation identified by next-generation sequencing techniques can be supported by recurrent disruptive variants in the same gene in phenotypically similar individuals. However, interpretation of novel variants in a specific gene in individuals with mild-moderate intellectual disability (ID) without recognizable syndromic features can be challenging and reverse phenotyping is often required. We describe 24 individuals with a de novo disease-causing variant in, or partial deletion of, the F-box only protein 11 gene (FBXO11, also known as VIT1 and PRMT9). FBXO11 is part of the SCF (SKP1-cullin-F-box) complex, a multi-protein E3 ubiquitin-ligase complex catalyzing the ubiquitination of proteins destined for proteasomal degradation. Twenty-two variants were identified by next-generation sequencing, comprising 2 in-frame deletions, 11 missense variants, 1 canonical splice site variant, and 8 nonsense or frameshift variants leading to a truncated protein or degraded transcript. The remaining two variants were identified by array-comparative genomic hybridization and consisted of a partial deletion of FBXO11. All individuals had borderline to severe ID and behavioral problems (autism spectrum disorder, attention-deficit/hyperactivity disorder, anxiety, aggression) were observed in most of them. The most relevant common facial features included a thin upper lip and a broad prominent space between the paramedian peaks of the upper lip. Other features were hypotonia and hyperlaxity of the joints. We show that de novo variants in FBXO11 cause a syndromic form of ID. The current series show the power of reverse phenotyping in the interpretation of novel genetic variances in individuals who initially did not appear to have a clear recognizable phenotype.

Whole-gene duplications and missense variants in the HUWE1 gene (NM_031407.6) have been reported in association with intellectual disability (ID). Increased gene dosage has been observed in males with non-syndromic mild to moderate ID with speech delay. Missense variants reported previously appear to be associated with severe ID in males and mild or no ID in obligate carrier females. Here, we report the largest cohort of patients with HUWE1 variants, consisting of 14 females and 7 males, with 15 different missense variants and one splice site variant. Clinical assessment identified common clinical features consisting of moderate to profound ID, delayed or absent speech, short stature with small hands and feet and facial dysmorphism consisting of a broad nasal tip, deep set eyes, epicanthic folds, short palpebral fissures, and a short philtrum. We describe for the first time that females can be severely affected, despite preferential inactivation of the affected X chromosome. Three females with the c.329 G  >  A p.Arg110Gln variant, present with a phenotype of mild ID, specific facial features, scoliosis and craniosynostosis, as reported previously in a single patient. In these females, the X inactivation pattern appeared skewed in favour of the affected transcript. In summary, HUWE1 missense variants may cause syndromic ID in both males and females.

Genotype-first combined with reverse phenotyping has shown to be a powerful tool in human genetics, especially in the era of next generation sequencing. This combines the identification of individuals with mutations in the same gene and linking these to consistent (endo)phenotypes to establish disease causality. We have performed a MIP (molecular inversion probe)-based targeted re-sequencing study in 3,275 individuals with intellectual disability (ID) to facilitate a genotype-first approach for 24 genes previously implicated in ID.Combining our data with data from a publicly available database, we confirmed 11 of these 24 genes to be relevant for ID. Amongst these, PHIP was shown to have an enrichment of disruptive mutations in the individuals with ID (5 out of 3,275). Through international collaboration, we identified a total of 23 individuals with PHIP mutations and elucidated the associated phenotype. Remarkably, all 23 individuals had developmental delay/ID and the majority were overweight or obese. Other features comprised behavioral problems (hyperactivity, aggression, features of autism and/or mood disorder) and dysmorphisms (full eyebrows and/or synophrys, upturned nose, large ears and tapering fingers). Interestingly, PHIP encodes two protein-isoforms, PHIP/DCAF14 and NDRP, each involved in neurodevelopmental processes, including E3 ubiquitination and neuronal differentiation. Detailed genotype-phenotype analysis points towards haploinsufficiency of PHIP/DCAF14, and not NDRP, as the underlying cause of the phenotype.Thus, we demonstrated the use of large scale re-sequencing by MIPs, followed by reverse phenotyping, as a constructive approach to verify candidate disease genes and identify novel syndromes, highlighted by PHIP haploinsufficiency causing an ID-overweight syndrome.

Burn-McKeown syndrome (BMKS) is a rare syndrome characterized by choanal atresia, prominent ears, abnormalities of the outer third of the lower eyelid, structural cardiac abnormalities, conductive and sensorineural hearing loss, and cleft lip. Recently, causative compound heterozygous variants were identified in TXNL4A. We analyzed an individual with clinical features of BMKS and her parents by whole-genome sequencing and identified compound heterozygous variants in TXNL4A (a novel splice site variant (c.258-2A>G, (p.?)) and a 34 bp promoter deletion (hg19 chr18:g.77748581_77748614del (type 1Δ) in the proband). Subsequently, we tested a cohort of 19 individuals with (mild) features of BMKS and 17 individuals with isolated choanal atresia for causative variants in TXNL4A by dideoxy-sequence analysis. In one individual with BMKS unrelated to the first family, we identified the identical compound heterozygous variants. In an individual with isolated choanal atresia, we found homozygosity for the same type 1Δ promoter deletion, whilst in two cousins from a family with choanal atresia and other minor anomalies we found homozygosity for a different deletion within the promoter (hg19 chr18: g.77748604_77748637del (type 2Δ)). Hence, we identified causative recessive variants in TXNL4A in two individuals with BMKS as well as in three individuals (from two families) with isolated choanal atresia.

Inherited eye disorders have a large clinical and genetic heterogeneity, which makes genetic diagnosis cumbersome. An exome-sequencing approach was developed in which data analysis was divided into two steps: the vision gene panel and exome analysis. In the vision gene panel analysis, variants in genes known to cause inherited eye disorders were assessed for pathogenicity. If no causative variants were detected and when the patient consented, the entire exome data was analyzed. A total of 266 Dutch patients with different types of inherited eye disorders, including inherited retinal dystrophies, cataract, developmental eye disorders and optic atrophy, were investigated. In the vision gene panel analysis (likely), causative variants were detected in 49% and in the exome analysis in an additional 2% of the patients. The highest detection rate of (likely) causative variants was in patients with inherited retinal dystrophies, for instance a yield of 63% in patients with retinitis pigmentosa. In patients with developmental eye defects, cataract and optic atrophy, the detection rate was 50, 33 and 17%, respectively. An exome-sequencing approach enables a genetic diagnosis in patients with different types of inherited eye disorders using one test. The exome approach has the same detection rate as targeted panel sequencing tests, but offers a number of advantages. For instance, the vision gene panel can be frequently and easily updated with additional (novel) eye disorder genes. Determination of the genetic diagnosis improved the clinical diagnosis, regarding the assessment of the inheritance pattern as well as future disease perspective.

Hearing impairment (HI) is genetically heterogeneous which hampers genetic counseling and molecular diagnosis. Testing of several single HI-related genes is laborious and expensive. In this study, we evaluate the diagnostic utility of whole-exome sequencing (WES) targeting a panel of HI-related genes. Two hundred index patients, mostly of Dutch origin, with presumed hereditary HI underwent WES followed by targeted analysis of an HI gene panel of 120 genes. We found causative variants underlying the HI in 67 of 200 patients (33.5%). Eight of these patients have a large homozygous deletion involving STRC, OTOA or USH2A, which could only be identified by copy number variation detection. Variants of uncertain significance were found in 10 patients (5.0%). In the remaining 123 cases, no potentially causative variants were detected (61.5%). In our patient cohort, causative variants in GJB2, USH2A, MYO15A and STRC, and in MYO6 were the leading causes for autosomal recessive and dominant HI, respectively. Segregation analysis and functional analyses of variants of uncertain significance will probably further increase the diagnostic yield of WES.

Autosomal dominant polycystic liver disease (ADPLD) is caused by variants in PRKCSH, SEC63, and LRP5, whereas autosomal dominant polycystic kidney disease is caused by variants in PKD1 and PKD2. Liver cyst development in these disorders is explained by somatic loss-of-heterozygosity (LOH) of the wild-type allele in the developing cyst. We hypothesize that we can use this mechanism to identify novel disease genes that reside in LOH regions. In this study, we aim to map abnormal genomic regions using high-density SNP microarrays to find novel PLD genes. We collected 46 cysts from 23 patients with polycystic or sporadic hepatic cysts, and analyzed DNA from those cysts using high-resolution microarray (n=24) or Sanger sequencing (n=22). We here focused on regions of homozygosity on the autosomes (>3.0 Mb) and large CNVs (>1.0 Mb). We found frequent LOH in PRKCSH (22/29) and PKD1/PKD2 (2/3) cysts of patients with known heterozygous germline variants in the respective genes. In the total cohort, 12/23 patients harbored abnormalities outside of familiar areas. In individual ADPLD cases, we identified germline events: a 2q13 complex rearrangement resulting in BUB1 haploinsufficiency, a 47XXX karyotype, chromosome 9q copy-number loss, and LOH on chromosome 3p. The latter region was overlapping with an LOH region identified in two other cysts. Unique germline and somatic abnormalities occur frequently in and outside of known genes underlying cysts. Each liver cyst has a unique genetic makeup. LOH driver gene BUB1 may imply germline causes of genetic instability in PLD.

To identify candidate genes for intellectual disability, we performed a meta-analysis on 2,637 de novo mutations, identified from the exomes of 2,104 patient-parent trios. Statistical analyses identified 10 new candidate ID genes: DLG4, PPM1D, RAC1, SMAD6, SON, SOX5, SYNCRIP, TCF20, TLK2 and TRIP12. In addition, we show that these genes are intolerant to nonsynonymous variation and that mutations in these genes are associated with specific clinical ID phenotypes.

Recently WAC was reported as a candidate gene for intellectual disability (ID) based on the identification of a de novo mutation in an individual with severe ID. WAC regulates transcription-coupled histone H2B ubiquitination and has previously been implicated in the 10p12p11 contiguous gene deletion syndrome. In this study, we report on 10 individuals with de novo WAC mutations which we identified through routine (diagnostic) exome sequencing and targeted resequencing of WAC in 2326 individuals with unexplained ID. All but one mutation was expected to lead to a loss-of-function of WAC. Clinical evaluation of all individuals revealed phenotypic overlap for mild ID, hypotonia, behavioral problems and distinctive facial dysmorphisms, including a square-shaped face, deep set eyes, long palpebral fissures, and a broad mouth and chin. These clinical features were also previously reported in individuals with 10p12p11 microdeletion syndrome. To investigate the role of WAC in ID, we studied the importance of the Drosophila WAC orthologue (CG8949) in habituation, a non-associative learning paradigm. Neuronal knockdown of Drosophila CG8949 resulted in impaired learning, suggesting that WAC is required in neurons for normal cognitive performance. In conclusion, we defined a clinically recognizable ID syndrome, caused by de novo loss-of-function mutations in WAC. Independent functional evidence in Drosophila further supported the role of WAC in ID. On the basis of our data WAC can be added to the list of ID genes with a role in transcription regulation through histone modification.

The Koolen-de Vries syndrome (KdVS; OMIM #610443), also known as the 17q21.31 microdeletion syndrome, is a clinically heterogeneous disorder characterised by (neonatal) hypotonia, developmental delay, moderate intellectual disability, and characteristic facial dysmorphism. Expressive language development is particularly impaired compared with receptive language or motor skills. Other frequently reported features include social and friendly behaviour, epilepsy, musculoskeletal anomalies, congenital heart defects, urogenital malformations, and ectodermal anomalies. The syndrome is caused by a truncating variant in the KAT8 regulatory NSL complex unit 1 (KANSL1) gene or by a 17q21.31 microdeletion encompassing KANSL1. Herein we describe a novel cohort of 45 individuals with KdVS of whom 33 have a 17q21.31 microdeletion and 12 a single-nucleotide variant (SNV) in KANSL1 (19 males, 26 females; age range 7 months to 50 years). We provide guidance about the potential pitfalls in the laboratory testing and emphasise the challenges of KANSL1 variant calling and DNA copy number analysis in the complex 17q21.31 region. Moreover, we present detailed phenotypic information, including neuropsychological features, that contribute to the broad phenotypic spectrum of the syndrome. Comparison of the phenotype of both the microdeletion and SNV patients does not show differences of clinical importance, stressing that haploinsufficiency of KANSL1 is sufficient to cause the full KdVS phenotype.

Cerebral visual impairment (CVI) is a major cause of low vision in children due to impairment in projection and/or interpretation of the visual input in the brain. Although acquired causes for CVI are well known, genetic causes underlying CVI are largely unidentified. DNAs of 25 patients with CVI and intellectual disability, but without acquired (eg, perinatal) damage, were investigated by whole-exome sequencing. The data were analyzed for de novo, autosomal-recessive, and X-linked variants, and subsequently classified into known, candidate, or unlikely to be associated with CVI. This classification was based on the Online Mendelian Inheritance in Man database, literature reports, variant characteristics, and functional relevance of the gene. After classification, variants in four genes known to be associated with CVI (AHDC1, NGLY1, NR2F1, PGAP1) in 5 patients (20%) were identified, establishing a conclusive genetic diagnosis for CVI. In addition, in 11 patients (44%) with CVI, variants in one or more candidate genes were identified (ACP6, AMOT, ARHGEF10L, ATP6V1A, DCAF6, DLG4, GABRB2, GRIN1, GRIN2B, KCNQ3, KCTD19, RERE, SLC1A1, SLC25A16, SLC35A2, SOX5, UFSP2, UHMK1, ZFP30). Our findings show that diverse genetic causes underlie CVI, some of which will provide insight into the biology underlying this disease process.

AIMP1/p43 is a multifunctional non-catalytic component of the multisynthetase complex. The complex consists of nine catalytic and three non-catalytic proteins, which catalyze the ligation of amino acids to their cognate tRNA isoacceptors for use in protein translation. To date, two allelic variants in the AIMP1 gene have been reported as the underlying cause of autosomal recessive primary neurodegenerative disorder. Here, we present two consanguineous families from Pakistan and Iran, presenting with moderate to severe intellectual disability, global developmental delay, and speech impairment without neurodegeneration. By the combination of homozygosity mapping and next generation sequencing, we identified two homozygous missense variants, p.(Gly299Arg) and p.(Val176Gly), in the gene AIMP1 that co-segregated with the phenotype in the respective families. Molecular modeling of the variants revealed deleterious effects on the protein structure that are predicted to result in reduced AIMP1 function. Our findings indicate that the clinical spectrum for AIMP1 defects is broader than witnessed so far.

Verheij syndrome, also called 8q24.3 microdeletion syndrome, is a rare condition characterized by ante- and postnatal growth retardation, microcephaly, vertebral anomalies, joint laxity/dislocation, developmental delay (DD), cardiac and renal defects and dysmorphic features. Recently, PUF60 (Poly-U Binding Splicing Factor 60 kDa), which encodes a component of the spliceosome, has been discussed as the best candidate gene for the Verheij syndrome phenotype, regarding the cardiac and short stature phenotype. To date, only one patient has been reported with a de novo variant in PUF60 that probably affects function (c.505C>T leading to p.(His169Tyr)) associated with DD, microcephaly, craniofacial and cardiac defects. Additional patients were required to confirm the pathogenesis of this association and further delineate the clinical spectrum. Here we report five patients with de novo heterozygous variants in PUF60 identified using whole exome sequencing. Variants included a splice-site variant (c.24+1G>C), a frameshift variant (p.(Ile136Thrfs*31)), two nonsense variants (p.(Arg448*) and p.(Lys301*)) and a missense change (p.(Val483Ala)). All six patients with a PUF60 variant (the five patients of the present study and the unique reported patient) have the same core facial gestalt as 8q24.3 microdeletions patients, associated with DD. Other findings include feeding difficulties (3/6), cardiac defects (5/6), short stature (5/6), joint laxity and/or dislocation (5/6), vertebral anomalies (3/6), bilateral microphthalmia and irido-retinal coloboma (1/6), bilateral optic nerve hypoplasia (2/6), renal anomalies (2/6) and branchial arch defects (2/6). These results confirm that PUF60 is a major driver for the developmental, craniofacial, skeletal and cardiac phenotypes associated with the 8q24.3 microdeletion.

Severe intellectual disability (ID) occurs in 0.5% of newborns and is thought to be largely genetic in origin. The extensive genetic heterogeneity of this disorder requires a genome-wide detection of all types of genetic variation. Microarray studies and, more recently, exome sequencing have demonstrated the importance of de novo copy number variations (CNVs) and single-nucleotide variations (SNVs) in ID, but the majority of cases remain undiagnosed. Here we applied whole-genome sequencing to 50 patients with severe ID and their unaffected parents. All patients included had not received a molecular diagnosis after extensive genetic prescreening, including microarray-based CNV studies and exome sequencing. Notwithstanding this prescreening, 84 de novo SNVs affecting the coding region were identified, which showed a statistically significant enrichment of loss-of-function mutations as well as an enrichment for genes previously implicated in ID-related disorders. In addition, we identified eight de novo CNVs, including single-exon and intra-exonic deletions, as well as interchromosomal duplications. These CNVs affected known ID genes more frequently than expected. On the basis of diagnostic interpretation of all de novo variants, a conclusive genetic diagnosis was reached in 20 patients. Together with one compound heterozygous CNV causing disease in a recessive mode, this results in a diagnostic yield of 42% in this extensively studied cohort, and 62% as a cumulative estimate in an unselected cohort. These results suggest that de novo SNVs and CNVs affecting the coding region are a major cause of severe ID. Genome sequencing can be applied as a single genetic test to reliably identify and characterize the comprehensive spectrum of genetic variation, providing a genetic diagnosis in the majority of patients with severe ID.

Gitelman syndrome (GS) is an autosomal recessive disorder characterized by hypokalemic metabolic alkalosis in conjunction with significant hypomagnesemia and hypocalciuria. The GS phenotype is caused by mutations in the solute carrier family 12, member 3 (SLC12A3) gene that encodes the thiazide-sensitive NaCl cotransporter (NCC). We analyzed DNA samples of 163 patients with a clinical suspicion of GS by direct sequencing of all 26 exons of the SLC12A3 gene. In total, 114 different mutations were identified, 31 of which have not been reported before. These novel variants include 3 deletions, 18 missense, 6 splice site and 4 nonsense mutations. We selected seven missense mutations to investigate their effect on NCC activity and plasma membrane localization by using the Xenopus laevis oocyte expression system. The Thr392Ile mutant did not display transport activity (probably class 2 mutation), while the Asn442Ser and Gln1030Arg NCC mutants showed decreased plasma membrane localization and consequently function, likely due to impaired trafficking (class 3 mutation). Even though the NaCl uptake was hampered for NCC mutants Glu121Asp, Pro751Leu, Ser475Cys and Tyr489His, the transporters reached the plasma membrane (class 4 mutation), suggesting an effect on NCC regulation or ion affinity. The present study shows the identification of 38 novel mutations in the SLC12A3 gene and provides insight into the mechanisms that regulate NCC.

In several laboratories, genome-wide array analysis has been implemented as the first tier diagnostic test for the identification of copy number changes in patients with mental retardation and/or congenital anomalies. The identification of a pathogenic copy number variant (CNV) is not only important to make a proper diagnosis but also to enable the accurate estimation of the recurrence risk to family members. Upon the identification of a de novo interstitial loss or gain, the risk recurrence is considered very low. However, this risk is 50% if one of the parents is carrier of a balanced insertional translocation (IT). The apparently de novo imbalance in a patient is then the consequence of the unbalanced transmission of a derivative chromosome involved in an IT. To determine the frequency with which insertional balanced translocations would be the origin of submicroscopic imbalances, we investigated the potential presence of an IT in a consecutive series of 477 interstitial CNVs, in which the parental origin has been tested by FISH, among 14,293 patients with developmental abnormalities referred for array. We demonstrate that ITs underlie ~2.1% of the apparently de novo, interstitial CNVs, indicating that submicroscopic ITs are at least sixfold more frequent than cytogenetically visible ITs. This risk estimate should be taken into account during counseling, and warrant parental and proband FISH testing wherever possible in patients with an apparently de novo, interstitial aberration.

Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.

Autosomal recessive mental retardation (AR-MR) may account for up to 25% of genetic mental retardation (MR). So far, mapping of AR-MR genes in consanguineous families has resulted in six nonsyndromic genes, whereas more than 2000 genes might contribute to AR-MR. We propose to use outbred families with multiple affected siblings for AR-MR gene identification. Homozygosity mapping in ten outbred families with affected brother-sister pairs using a 250 K single nucleotide polymorphism array revealed on average 57 homozygous regions over 1 Mb in size per affected individual (range 20-74). Of these, 21 homozygous regions were shared between siblings on average (range 8-36). None of the shared regions of homozygosity (SROHs) overlapped with the nonsyndromic genes. A total of 13 SROHs had an overlap with previously reported loci for AR-MR, namely with MRT8, MRT9, MRT10 and MRT11. Among these was the longest observed SROH of 11.0 Mb in family ARMR1 on chromosome 19q13, which had 2.9 Mb (98 genes) in common with the 5.4 Mb MRT11 locus (195 genes). These data support that homozygosity mapping in outbred families may contribute to identification of novel AR-MR genes.

The oxidative phosphorylation (OXPHOS) system is under control of both the mitochondrial and the nuclear genomes; 13 subunits are synthesized by the mitochondrial translation machinery. We report a patient with Cornelia de Lange-like dysmorphic features, brain abnormalities and hypertrophic cardiomyopathy, and studied the genetic defect responsible for the combined OXPHOS complex I, III and IV deficiency observed in fibroblasts. The combination of deficiencies suggested a primary defect associated with the synthesis of mitochondrially encoded OXPHOS subunits. Analysis of mitochondrial protein synthesis revealed a marked impairment in mitochondrial translation. Homozygosity mapping and sequence analysis of candidate genes revealed a homozygous mutation in MRPS22, a gene encoding a mitochondrial ribosomal small subunit protein. The mutation predicts a Leu215Pro substitution at an evolutionary conserved site. Mutations in genes implicated in Cornelia de Lange syndrome or copy number variations were not found. Transfection of patient fibroblasts, in which MRPS22 was undetectable, with the wild-type MRPS22 cDNA restored the amount and activity of OXPHOS complex IV, as well as the 12S rRNA transcript level to normal values. These findings demonstrate the pathogenicity of the MRPS22 mutation and stress the significance of mutations in nuclear genes, including genes that have no counterparts in lower species like bacteria and yeast, for mitochondrial translation defects.

The genomic architecture of the 10q22q23 region is characterised by two low-copy repeats (LCRs3 and 4), and deletions in this region appear to be rare. We report the clinical and molecular characterisation of eight novel deletions and six duplications within the 10q22.3q23.3 region. Five deletions and three duplications occur between LCRs3 and 4, whereas three deletions and three duplications have unique breakpoints. Most of the individuals with the LCR3-4 deletion had developmental delay, mainly affecting speech. In addition, macrocephaly, mild facial dysmorphisms, cerebellar anomalies, cardiac defects and congenital breast aplasia were observed. For congenital breast aplasia, the NRG3 gene, known to be involved in early mammary gland development in mice, is a putative candidate gene. For cardiac defects, BMPR1A and GRID1 are putative candidate genes because of their association with cardiac structure and function. Duplications between LCRs3 and 4 are associated with variable phenotypic penetrance. Probands had speech and/or motor delays and dysmorphisms including a broad forehead, deep-set eyes, upslanting palpebral fissures, a smooth philtrum and a thin upper lip. In conclusion, duplications between LCRs3 and 4 on 10q22.3q23.2 may lead to a distinct facial appearance and delays in speech and motor development. However, the phenotypic spectrum is broad, and duplications have also been found in healthy family members of a proband. Reciprocal deletions lead to speech and language delay, mild facial dysmorphisms and, in some individuals, to cerebellar, breast developmental and cardiac defects.

The clinical use of array comparative genomic hybridization in the evaluation of patients with multiple congenital anomalies and/or mental retardation has recently led to the discovery of a number of novel microdeletion and microduplication syndromes. We present four male patients with overlapping molecularly defined de novo microdeletions of 16q24.3. The clinical features observed in these patients include facial dysmorphisms comprising prominent forehead, large ears, smooth philtrum, pointed chin and wide mouth, variable cognitive impairment, autism spectrum disorder, structural anomalies of the brain, seizures and neonatal thrombocytopenia. Although deletions vary in size, the common region of overlap is only 90 kb and comprises two known genes, Ankyrin Repeat Domain 11 (ANKRD11) (MIM 611192) and Zinc Finger 778 (ZNF778), and is located approximately 10 kb distally to Cadherin 15 (CDH15) (MIM 114019). This region is not found as a copy number variation in controls. We propose that these patients represent a novel and distinctive microdeletion syndrome, characterized by autism spectrum disorder, variable cognitive impairment, facial dysmorphisms and brain abnormalities. We suggest that haploinsufficiency of ANKRD11 and/or ZNF778 contribute to this phenotype and speculate that further investigation of non-deletion patients who have features suggestive of this 16q24.3 microdeletion syndrome might uncover other mutations in one or both of these genes.

Six submicroscopic deletions comprising chromosome band 2q23.1 in patients with severe mental retardation (MR), short stature, microcephaly and epilepsy have been reported, suggesting that haploinsufficiency of one or more genes in the 2q23.1 region might be responsible for the common phenotypic features in these patients. In this study, we report the molecular and clinical characterisation of nine new 2q23.1 deletion patients and a clinical update on two previously reported patients. All patients were mentally retarded with pronounced speech delay and additional abnormalities including short stature, seizures, microcephaly and coarse facies. The majority of cases presented with stereotypic repetitive behaviour, a disturbed sleep pattern and a broad-based gait. These features led to the initial clinical impression of Angelman, Rett or Smith-Magenis syndromes in several patients. The overlapping 2q23.1 deletion region in all 15 patients comprises only one gene, namely, MBD5. Interestingly, MBD5 is a member of the methyl CpG-binding domain protein family, which also comprises MECP2, mutated in Rett's syndrome. Another gene in the 2q23.1 region, EPC2, was deleted in 12 patients who had a broader phenotype than those with a deletion of MBD5 only. EPC2 is a member of the polycomb protein family, involved in heterochromatin formation and might be involved in causing MR. Patients with a 2q23.1 microdeletion present with a variable phenotype and the diagnosis should be considered in mentally retarded children with coarse facies, seizures, disturbed sleeping patterns and additional specific behavioural problems.

Identification of genetic copy number changes in glial tumors is of importance in the context of improved/refined diagnostic, prognostic procedures and therapeutic decision-making. In order to detect recurrent genomic copy number changes that might play a role in glioma pathogenesis and/or progression, we characterized 25 primary glioma cell lines including 15 non glioblastoma (non GBM) (I-III WHO grade) and 10 GBM (IV WHO grade), by array comparative genomic hybridization, using a DNA microarray comprising approx. 3500 BACs covering the entire genome with a 1 Mb resolution and additional 800 BACs covering chromosome 19 at tiling path resolution. Combined evaluation by single clone and whole chromosome analysis plus 'moving average (MA) approach' enabled us to confirm most of the genetic abnormalities previously identified to be associated with glioma progression, including +1q32, +7, -10, -22q, PTEN and p16 loss, and to disclose new small genomic regions, some correlating with grade malignancy. Grade I-III gliomas exclusively showed losses at 3p26 (53%), 4q13-21 (33%) and 7p15-p21 (26%), whereas only GBMs exhibited 4p16.1 losses (40%). Other recurrent imbalances, such as losses at 4p15, 5q22-q23, 6p23-25, 12p13 and gains at 11p11-q13, were shared by different glioma grades. Three intervals with peak of loss could be further refined for chromosome 10 by our MA approach. Data analysis of full-coverage chromosome 19 highlighted two main regions of copy number gain, never described before in gliomas, at 19p13.11 and 19q13.13-13.2. The well-known 19q13.3 loss of heterozygosity area in gliomas was not frequently affected in our cell lines. Genomic hotspot detection facilitated the identification of small intervals resulting in positional candidate genes such as PRDM2 (1p36.21), LRP1B (2q22.3), ADARB2 (10p15.3), BCCIP (10q26.2) and ING1 (13q34) for losses and ECT2 (3q26.3), MDK, DDB2, IG20 (11p11.2) for gains. These data increase our current knowledge about cryptic genetic changes in gliomas and may facilitate the further identification of novel genetic elements, which may provide us with molecular tools for the improved diagnostics and therapeutic decision-making in these tumors.