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Inversions are balanced structural variants that often remain undetected in genetic diagnostics. We present a female proband with a de novo Chromosome 15 paracentric inversion, disrupting MEIS2 and NUSAP1. The inversion was detected by short-read genome sequencing and confirmed with adaptive long-read sequencing. The breakpoint junction analysis revealed a 96 bp (bp) deletion and an 18 bp insertion in the two junctions, suggesting that the rearrangement arose through a replicative error. Transcriptome sequencing of cultured fibroblasts revealed normal MEIS2 levels and 0.61-fold decreased expression of NUSAP1. Furthermore, three fusion transcripts were detected and confirmed by Sanger sequencing. Heterozygous loss of MEIS2 (MIM# 600987) is associated with a cleft palate, heart malformations, and intellectual impairment, which overlap with the clinical symptoms observed in the proband. The observed fusion transcripts are likely non-functional, and MEIS2 haploinsufficiency is the likely disease causative mechanism. Altogether, this study's findings illustrate the importance of including inversions in rare disease diagnostic testing and highlight the value of long read sequencing for the validation and characterization of such variants., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The introduction of whole-exome sequencing into the Pediatric Genetics clinic has increased the identification of novel genes associated with neurodevelopmental disorders and congenital anomalies. This agnostic approach has shed light on multiple proteins and pathways not previously known to be associated with disease. Here we report eight subjects from six families with predicted loss of function variants in ZNF462, a zinc-finger protein of unknown function. These individuals have overlapping phenotypes that include ptosis, metopic ridging, craniosynostosis, dysgenesis of the corpus callosum, and developmental delay. We propose that ZNF462 plays an important role in embryonic development, and is associated with craniofacial and neurodevelopmental abnormalities.

Although de novo missense mutations have been predicted to account for more cases of autism than gene-truncating mutations, most research has focused on the latter. We identified the properties of de novo missense mutations in patients with neurodevelopmental disorders (NDDs) and highlight 35 genes with excess missense mutations. Additionally, 40 amino acid sites were recurrently mutated in 36 genes, and targeted sequencing of 20 sites in 17,688 patients with NDD identified 21 new patients with identical missense mutations. One recurrent site substitution (p.A636T) occurs in a glutamate receptor subunit, GRIA1. This same amino acid substitution in the homologous but distinct mouse glutamate receptor subunit Grid2 is associated with Lurcher ataxia. Phenotypic follow-up in five individuals with GRIA1 mutations shows evidence of specific learning disabilities and autism. Overall, we find significant clustering of de novo mutations in 200 genes, highlighting specific functional domains and synaptic candidate genes important in NDD pathology.

We report on seven novel patients with a submicroscopic 22q12 deletion. The common phenotype constitutes a contiguous gene deletion syndrome on chromosome 22q12.1q12.2, featuring NF2-related schwannoma of the vestibular nerve, corpus callosum agenesis and palatal defects. Combining our results with the literature, eight patients are recorded with palatal defects in association with haploinsufficiency of 22q12.1, including the MN1 gene. These observations, together with the mouse expression data and the finding of craniofacial malformations including cleft palate in a Mn1-knockout mouse model, suggest that this gene is a candidate gene for cleft palate in humans.

KAT6B sequence variants have been identified previously in both patients with the Say-Barber-Biesecker type of blepharophimosis mental retardation syndromes (SBBS) and in the more severe genitopatellar syndrome (GPS). We report on the findings in a previously unreported group of 57 individuals with suggestive features of SBBS or GPS. Likely causative variants have been identified in 34/57 patients and were commonly located in the terminal exons of KAT6B. Of those where parental samples could be tested, all occurred de novo. Thirty out of thirty-four had truncating variants, one had a missense variant and the remaining three had the same synonymous change predicted to affect splicing. Variants in GPS tended to occur more proximally to those in SBBS patients, and genotype/phenotype analysis demonstrated significant clinical overlap between SBBS and GPS. The de novo synonymous change seen in three patients with features of SBBS occurred more proximally in exon 16. Statistical analysis of clinical features demonstrated that KAT6B variant-positive patients were more likely to display hypotonia, feeding difficulties, long thumbs/great toes and dental, thyroid and patella abnormalities than KAT6B variant-negative patients. The few reported patients with KAT6B haploinsufficiency had a much milder phenotype, though with some features overlapping those of SBBS. We report the findings in a previously unreported patient with a deletion of the KAT6B gene to further delineate the haploinsufficiency phenotype. The molecular mechanisms giving rise to the SBBS and GPS phenotypes are discussed.

Deletions involving 17q21-q24 have been identified previously to result in two clinically recognizable contiguous gene deletion syndromes: 17q21.31 and 17q23.1-q23.2 microdeletion syndromes. Although deletions involving 17q22 have been reported in the literature, only four of the eight patients reported were identified by array-comparative genomic hybridization (array-CGH) or flourescent in situ hybridization. Here, we describe five new patients with 1.8-2.5-Mb microdeletions involving 17q22 identified by array-CGH. We also present one patient with a large karyotypically visible deletion involving 17q22, fine-mapped to ~8.2 Mb using array-CGH. We show that the commonly deleted region in our patients spans 0.24 Mb and two genes; NOG and C17ORF67. The function of C17ORF67 is not known, whereas Noggin, the product of NOG, is essential for correct joint development. In common with the 17q22 patients reported previously, the disease phenotype of our patients includes intellectual disability, attention deficit hyperactivity disorder, conductive hearing loss, visual impairment, low set ears, facial dysmorphology and limb anomalies. All patients displayed NOG-related bone and joint features, including symphalangism and facial dysmorphology. We conclude that these common clinical features indicate a novel clinically recognizable, 17q22 contiguous microdeletion syndrome.

The introduction of array CGH in clinical diagnostics has led to the discovery of many new microdeletion/microduplication syndromes. Most of them are rare and often present with a variable range of clinical anomalies. In this study we report three patients with a de novo overlapping microdeletion of chromosome bands 12q15q21.1. The deletions are ∼2.5 Mb in size, with a 1.34-Mb common deleted region containing six RefSeq genes. All three patients present with learning disability or developmental delay, nasal speech and hypothyroidism. In this paper we will further elaborate on the genotype-phenotype correlation associated with this deletion and compare our patients with previously reported cases.

The distal region on the short arm of chromosome 9 is of special interest for scientists interested in sex development as well as in the clinical phenotype of patients with the 9p deletion syndrome, characterized by mental retardation, trigonocephaly and other dysmorphic features. Specific genes responsible for different aspects of the phenotype have not been identified. Distal 9p deletions have also been reported in patients with 46,XY sex reversal, with or without 9p deletion syndrome. Within this region the strongest candidates for the gonadal dysgenesis phenotype are the DMRT genes; however, the genetic mechanism is not clear yet. Multiple ligation-dependent probe amplification represents a useful technique to evaluate submicroscopic interstitial or distal deletions that would help the definition of the minimal sex reversal region on 9p and could lead to the identification of gene(s) responsible of the 46,XY gonadal disorders of sex development (DSD). We designed a synthetic probe set that targets genes within the 9p23-9p24.3 region and analyzed a group of XY patients with impaired gonadal development. We characterized a deletion distal to the DMRT genes in a patient with isolated 46,XY gonadal DSD and narrowed down the breakpoint in a patient with a 46,XY del(9)(p23) karyotype with gonadal DSD and mild symptoms of 9p deletion syndrome. The results are compared with other patients described in the literature, and new aspects of sex reversal and the 9p deletion syndrome candidate regions are discussed.

Genome-wide analysis of DNA copy-number changes using microarray-based technologies has enabled the detection of de novo cryptic chromosome imbalances in approximately 10% of individuals with mental retardation. So far, the majority of these submicroscopic microdeletions/duplications appear to be unique, hampering clinical interpretation and genetic counselling. We hypothesised that the genomic regions involved in these de novo submicroscopic aberrations would be candidates for recurrent copy-number changes in individuals with mental retardation. To test this hypothesis, we used multiplex ligation-dependent probe amplification (MLPA) to screen for copy number changes at eight genomic candidate regions in a European cohort of 710 individuals with idiopathic mental retardation. By doing so, we failed to detect additional submicroscopic rearrangements, indicating that the anomalies tested are non-recurrent in this cohort of patients. The break points flanking the candidate regions did not contain low copy repeats and/or sequence similarities, thus providing an explanation for its non-recurrent nature. On the basis of these data, we propose that the use of genome-wide microarrays is indicated when testing for copy-number changes in individuals with idiopathic mental retardation.

Cornelia de Lange syndrome (CdLS; OMIM 122470) is a rare multiple congenital anomaly/mental retardation syndrome characterized by distinctive dysmorphic facial features, severe growth and developmental delay and abnormalities of the upper limbs. About 50% of CdLS patients have been found to have heterozygous mutations in the NIPBL gene and a few cases were recently found to be caused by mutations in the X-linked SMC1L1 gene. We performed a mutation screening of all NIPBL coding exons by direct sequencing in 11 patients (nine sporadic and two familial cases) diagnosed with CdLS in Sweden and detected mutations in seven of the cases. All were de novo, and six of the mutations have not been previously described. Four patients without identifiable NIPBL mutations were subsequently subjected to multiplex ligation-dependent probe amplification analysis to exclude whole exon deletions/duplications of NIPBL. In addition, mutation analysis of the 5' untranslated region (5' UTR) of NIPBL was performed. Tiling resolution array comparative genomic hybridization analysis was carried out on these four patients to detect cryptic chromosome imbalances and in addition the boys were screened for SMC1L1 mutations. We found a de novo 9p duplication with a size of 0.6 Mb in one of the patients with a CdLS-like phenotype but no mutations were detected in SMC1L1. So far, two genes (NIPBL and SMC1L1) have been identified causing CdLS or CdLS-like phenotypes. However, in a considerable proportion of individuals demonstrating the CdLS phenotype, mutations in any of these two genes are not found and other potential loci harboring additional CdLS-causing genes should be considered.

Kabuki syndrome (KS) is a rare multiple congenital anomaly/mental retardation syndrome. It is characterized by a distinct facial appearance, mental retardation, postnatal growth retardation, skeletal anomalies, unusual dermatoglyphics and fetal fingertip pads. It has previously been speculated that KS is caused by a microdeletion or duplication. In a recent report, an interstitial microduplication of 8p22-23.1 was presented in several cases with this disorder. We investigated 10 Caucasian patients diagnosed with KS by fluorescence in situ hybridization and microsatellite markers located on 8p22-23.1. Using the same clones that were previously reported to be duplicated on chromosome 8p, we could exclude the duplication in all our patients. In addition, we performed a genome-wide screening on this group of patients using array-based comparative genomic hybridization containing BAC clones spaced at approximately 1 Mb intervals across the genome and could not find any evidence for gene dose alterations. The characteristics of KS are variable, a fact that complicates the diagnosis of this disorder. It is possible that we will find genetic heterogeneity among Kabuki patients, and therefore it is unlikely that all patients have an interstitial 8p duplication. We conclude that the etiology of KS remains to be solved and further genetic studies are necessary to delineate its genetic cause.

An increasing body of evidence indicates that submicroscopic gene dose alterations may cause mental impairment and malformations. During the last decade, comparative genomic hybridization (CGH) has become a useful tool in the detection and mapping of chromosome aberrations. Modifications of CGH with increased resolution down to 3-5 Mb have been reported and CGH is now offered as a diagnostic procedure in the evaluation of patients with idiopathic mental retardation (MR). In order to increase the resolution, we modified the CGH protocol using freshly prepared high-quality metaphase slides and chemical labeling, and tested the method on a set of patients with well-defined submicroscopic chromosome abnormalities with confirmed size 1.3-20.5 Mb. Subsequently, a completely blinded test was performed to compare the performance of the chemical labeling CGH to the commercially available HR-CGH. Using the two different CGH methods, we were able to detect chromosome imbalances down to 2-3 Mb approximately. The HR-CGH method detected all aberrations >6 Mb and a few smaller, while the modified CGH method was able to detect all but three aberrations >1.8 Mb. The modified CGH method was superior in the detection of terminal imbalances, while the HR-CGH software was more successful in the detection of imbalances located very close to the centromeric regions. In conclusion, the resolution of metaphase CGH may be as high as 2-3 Mb but is most likely depending on the chromosomal region involved, a clear limitation when used as a screening method for chromosome aberrations in patients with idiopathic MR.

Several cases with microscopically visible, terminal 6p deletions have been described, and a distinct clinical phenotype has emerged, including developmental delay, congenital heart malformations, ocular abnormalities, hearing loss and a characteristic facial appearance. We report a patient with a submicroscopic 6p deletion, detected by subtelomeric screening using fluorescence in situ hybridisation. This girl presented with typical facial dysmorphic features, hearing impairment, malformation of the anterior eye segment, an ASD and severe language impairment. However, her cognitive functions were within the normal range. Detailed FISH analysis with 20 BAC probes covering the distal 6p25 region estimated the size of the terminal deletion to 2.1 Mb, and thus this case narrows down the critical region for the 6p phenotype. The forkhead transcription factor gene FOXC1, involved in a spectrum of anterior eye chamber disorders, is deleted in this patient, together with several characterised and putative genes with yet unknown function.