Differential alternative splicing analysis links variation in ZRSR2 to a novel type of oral-facial-digital syndrome.

PURPOSE: Oral-facial-digital (OFD) syndromes are genetically heterogeneous developmental disorders, caused by pathogenic variants in genes involved in primary cilia formation and function. We identified a previously undescribed type of OFD with brain anomalies, ranging from alobar holoprosencephaly to pituitary anomalies, in 6 unrelated families. METHODS: Exome sequencing of affected probands was supplemented with alternative splicing analysis in patient and control lymphoblastoid and fibroblast cell lines, and primary cilia structure analysis in patient fibroblasts. RESULTS: In 1 family with 2 affected males, we identified a germline variant in the last exon of ZRSR2, NM_005089.4:c.1211_1212del NP_005080.1:p.(Gly404GlufsTer23), whereas 7 affected males from 5 unrelated families were hemizygous for the ZRSR2 variant NM_005089.4:c.1207_1208del NP_005080.1:p.(Arg403GlyfsTer24), either occurring de novo or inherited in an X-linked recessive pattern. ZRSR2, located on chromosome Xp22.2, encodes a splicing factor of the minor spliceosome complex, which recognizes minor introns, representing 0.35% of human introns. Patient samples showed significant enrichment of minor intron retention. Among differentially spliced targets are ciliopathy-related genes, such as TMEM107 and CIBAR1. Primary fibroblasts containing the NM_005089.4:c.1207_1208del ZRSR2 variant had abnormally elongated cilia, confirming an association between defective U12-type intron splicing, OFD and abnormal primary cilia formation. CONCLUSION: We introduce a novel type of OFD associated with elongated cilia and differential splicing of minor intron-containing genes due to germline variation in ZRSR2.

Genotype-phenotype correlations of UBA2 mutations in patients with ectrodactyly.

Interstitial 19q13.11 deletions are associated with ectrodactyly, which has recently been linked to loss-of-function of the UBA2 gene. We report a boy with a de novo frameshift mutation in UBA2 (c.612delA (p.(Glu205Lysfs*63)), presenting with ectrodactyly of the feet associated with learning difficulties and minor physical anomalies. We review genotype-phenotype correlations in patients with chromosomal 19q13.11 microdeletions compared to those with intragenic UBA2 mutations.

Challenges in molecular diagnosis of X-linked Intellectual disability.

BACKGROUND: Intellectual disability (ID) affects 1-3% of the Western population and is heterogeneous in origin. Mutations in X-linked genes represent 5-10% of ID in males. Fragile X syndrome, due to the silencing of the FMR1 gene, is the most common form of ID, with a prevalence of around 1:5000 males. Females are usually non- or mildly affected carriers, and in a few rare cases, the only gender affected. Array comparative genome hybridization (aCGH) and next-generation sequencing (NGS) have dramatically changed the nature of human genome analysis leading to the identification of new X-linked intellectual disability syndromes and disease-causing genes. SOURCES OF DATA: Original papers, reviews, guidelines and experiences of the diagnostic laboratories. AREAS OF AGREEMENT: Family history and clinical examination still are essential to choose the appropriate diagnostic tests, including, a disease-specific genetic test, aCGH or FMR1 molecular analysis. If negative, NGS approaches like well-defined gene panels, whole exome, or even whole genome sequencing, are increasingly being used, improving diagnostics and leading to the identification of novel disease mechanisms. AREAS OF CONTROVERSY: The main challenge in the era of NGS is filtering and interpretation of the data generated by the analysis of a single individual. In X-linked cases, assessing pathogenicity is particularly challenging, even more when the variant is found to be inherited from a healthy carrier mother or when a heterozygous X-linked mutation is found in an impaired female. GROWING POINTS: At present, variant interpretation remains a challenging task, especially in X-linked disorders. We review the main difficulties and propose a comprehensive overview that might aid in variant interpretation. Establishing a genetic diagnosis facilitates counseling and allows better delineation of clinical phenotypes. AREAS TIMELY FOR DEVELOPING RESEARCH: To improve variant interpretation, there is need to refine in silico predictions with specific criteria for each gene, and to develop cost-effective functional tools, which can be easily transferred to diagnostics.

Next-generation sequencing in prenatal setting: Some examples of unexpected variant association.

The application of next-generation sequencing to fetal pathology has proved to increase the diagnostic yield in fetuses with abnormal ultrasounds. We retrospectively reviewed genetic data of 30 selected cases studied through targeted resequencing of OMIM genes. In our experience, clinical data proved to be essential to support diagnostic reasoning and enhance variants' assessment. The molecular diagnosis was reached in 19/30 (63%) cases. Only in 7/19 cases the molecular diagnosis confirmed the initial diagnostic hypothesis, showing the relevance of the genotype-first approach. According to the genotype-phenotype correlation, we were able to divide the solved cases into three groups: i) the correlation is well established but it was missed due to lack of specificity, unusual presentation or recent description; ii) the clinical presentation is much more severe than currently known for the underlying condition; iii) the correlation does not recapitulate the entire phenotype, possibly due to the fetal presentation or multiple coexisting conditions. Moreover, we found a higher proportion of recessive diagnosis in abnormal fetuses compared to cohorts of individuals with developmental delay. Our findings suggest that fetal pathology may be enriched in rare alleles and/or in unusual combinations, counter-selected in postnatal genomes and thus contributing to both phenotypic extremeness and atypical presentation.

A genotype-first approach identifies an intellectual disability-overweight syndrome caused by PHIP haploinsufficiency.

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.

Microdeletion of the escape genes KDM5C and IQSEC2 in a girl with severe intellectual disability and autistic features.

Intellectual disability (ID) is a very heterogeneous disorder with over 100 ID genes located on the X chromosome alone. Of these, KDM5C and IQSEC2 are located adjacent to each other at the Xp11.22 locus. While mutations in either of these genes are associated with severe ID in males, female carriers are mostly unaffected. Here, we report on a female patient with severe ID and autistic features carrying a de novo 0.4 Mb deletion containing six coding genes including KDM5C and IQSEC2. X-inactivation analysis revealed skewing in a lymphocyte-derived cell line from this patient with preferential inactivation of the mutant X chromosome. As the brain-expressed KDM5C and IQSEC2 genes escape X-inactivation, deletion of these alleles could still be detrimental despite skewing of X-inactivation. Indeed, mutations in either of both genes have been reported in a few female ID patients. Expression analysis in the patients' cell line revealed decreased KDM5C mRNA levels compared to female controls. IQSEC2 levels could not be compared due to very low expression in blood. Overall, our data suggest that heterozygous loss-of-function of the escape genes KDM5C and/or IQSEC2 can contribute to severe ID in female patients and should be taken into account in diagnostics.

De novo MECP2 duplications in two females with intellectual disability and unfavorable complete skewed X-inactivation.

Xq28 microduplications of MECP2 are a prominent cause of a severe syndromic form of intellectual disability (ID) in males. Females are usually unaffected through near to complete X-inactivation of the aberrant X chromosome (skewing). In rare cases, affected females have been described due to random X-inactivation. Here, we report on two female patients carrying de novo MECP2 microduplications on their fully active X chromosomes. Both patients present with ID and additional clinical features. Mono-allelic expression confirmed complete skewing of X-inactivation. Consequently, significantly enhanced MECP2 mRNA levels were observed. We hypothesize that the cause for the complete skewing is due to a more harmful mutation on the other X chromosome, thereby forcing the MECP2 duplication to become active. However, we could not unequivocally identify such a second mutation by array-CGH or exome sequencing. Our data underline that, like in males, increased MECP2 dosage in females can contribute to ID too, which should be taken into account in diagnostics.

Evidence for increased SOX3 dosage as a risk factor for X-linked hypopituitarism and neural tube defects.

Genomic duplications of varying lengths at Xq26-q27 involving SOX3 have been described in families with X-linked hypopituitarism. Using array-CGH we detected a 1.1 Mb microduplication at Xq27 in a large family with three males suffering from X-linked hypopituitarism. The duplication was mapped from 138.7 to 139.8 Mb, harboring only two annotated genes, SOX3 and ATP11C, and was shown to be a direct tandem copy number gain. Unexpectedly, the microduplication did not fully segregate with the disease in this family suggesting that SOX3 duplications have variable penetrance for X-linked hypopituitarism. In the same family, a female fetus presenting with a neural tube defect was also shown to carry the SOX3 copy number gain. Since we also demonstrated increased SOX3 mRNA levels in amnion cells derived from an unrelated t(X;22)(q27;q11) female fetus with spina bifida, we propose that increased levels of SOX3 could be a risk factor for neural tube defects.

Ubiquitin ligase HUWE1 regulates axon branching through the Wnt/ß-catenin pathway in a Drosophila model for intellectual disability.

We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN) was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh) by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of ß-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/ß-catenin pathway possibly by enhancing the degradation of dsh.

The mitochondrial solute carrier SLC25A5 at Xq24 is a novel candidate gene for non-syndromic intellectual disability.

Loss-of-function mutations in several different neuronal pathways have been related to intellectual disability (ID). Such mutations often are found on the X chromosome in males since they result in functional null alleles. So far, microdeletions at Xq24 reported in males always have been associated with a syndromic form of ID due to the loss of UBE2A. Here, we report on overlapping microdeletions at Xq24 that do not include UBE2A or affect its expression, in patients with non-syndromic ID plus some additional features from three unrelated families. The smallest region of overlap, confirmed by junction sequencing, harbors two members of the mitochondrial solute carrier family 25, SLC25A5 and SLC25A43. However, identification of an intragenic microdeletion including SLC25A43 but not SLC25A5 in a healthy boy excluded a role for SLC25A43 in cognition. Therefore, our findings point to SLC25A5 as a novel gene for non-syndromic ID. This highly conserved gene is expressed ubiquitously with high levels in cortex and hippocampus, and a presumed role in mitochondrial exchange of ADP/ATP. Our data indicate that SLC25A5 is involved in memory formation or establishment, which could add mitochondrial processes to the wide array of pathways that regulate normal cognitive functions.

Promoting role of cholecystokinin 2 receptor (CCK2R) in gastrointestinal stromal tumour pathogenesis.

The cholecystokinin 2 receptor (CCK2R/CCKBR) is expressed in gastrointestinal stromal tumours (GISTs). We sought to investigate the role of CCK2R in GIST pathogenesis. Molecular characterization of CCK2R was performed on a heterogeneous cohort of 50 GISTs. In addition, CCK2R expression was evaluated by immunohistochemistry (IHC), using tissue microarray (TMA) containing 292 GISTs, two cases of hyperplasia of interstitial Cajal's cells (ICC) and six gastric microscopic GISTs. Mono-allelic loss of the CCK2R/11p15 allele was identified in 13.7% of GISTs, having no impact on the level of CCK2R transcript expression. No CCK2R mutations were found. The CCK2Ri4sv, CCK2R splice variant with retention of intron 4 was detected in six of 20 tumours analysed. Wild-type CCK2R transcripts were commonly expressed (57.1% of cases) and this expression was highly correlated with gastric primary site of GISTs (p < 0.001). At the protein level, expression of CCK2R in incidental ICC hyperplasia and early stages of gastric GIST development was documented, and its gastric association was confirmed on GIST-TMA by IHC. To explore the in vivo effect of CCK2R activation on tumour growth, gastrin versus placebo was administered intraperitoneally in nude mice carrying human GIST xenografts. The tumour volume was followed for 10 weeks. The effect of this stimulation on tumour cell proliferation/apoptosis was assessed by IHC and KIT/PKC-θ signalling was evaluated by western blotting (WB). In vivo experiments showed a two-fold increase in the volume of tumours which were exposed to gastrin in comparison with non-exposed controls (p = 0.03), with a significant increase in mitotic activity (p = 0.04) and Ki-67 proliferation index (p = 0.008). By WB, gastrin stimulation resulted in hyper-activation of KIT and PKC-θ kinases, and in evident PI3K-AKT pathway over-activation. Our results indicate a promoting role of CCK2R on GIST tumourigenesis, particularly in tumours of gastric origin.

Congenital heart defects in a novel recurrent 22q11.2 deletion harboring the genes CRKL and MAPK1.

The proximal region of the long arm of chromosome 22 is rich in low copy repeats (LCR). Non-allelic homologous recombination (NAHR) between these substrates explains the high prevalence of recurrent rearrangements within this region. We have performed array comparative genomic hybridization in a normally developing girl with growth delay, microcephaly, and truncus arteriosus, and have identified a novel recurrent 22q11 deletion that spans LCR22-4 and partially affects the common 22q11.2 deletion syndrome and the distal 22q11 deletion syndrome. This deletion is atypical as it did not occur by NAHR between any of the major LCRs found on 22q11.2. However, the breakpoint containing regions coincide with highly homologous regions. An identical imbalance was reported previously in a patient with striking phenotypic similarity. Computational gene prioritization methods and biological evidence denote the genes CRKL and MAPK1 as the highest ranking candidates for causing congenital heart disease within the deleted region.

BMPR1A is a candidate gene for congenital heart defects associated with the recurrent 10q22q23 deletion syndrome.

Congenital heart defects (CHD) are associated with the recurrent 10q22q23 deletion syndrome and with partially overlapping distal 10q23.2.q23.31 microdeletions. We report on a de novo intragenic deletion of the BMPR1A gene in a normally developing adolescent boy with short stature, delayed puberty, facial dysmorphism and an atrioventricular septal defect. Based on this finding, complemented with computational prioritization data and molecular evidence in literature, the critical region for CHD on 10q23 can be downsized to a single gene, BMPR1A. Although loss-of-function mutations in BMPR1A typically result in juvenile polyposis syndrome, none of the patients with the typical 10q22q23 microdeletion syndrome, comprising this gene, were reported to have juvenile polyposis thus far. We reason that, even in the absence of juvenile polyposis syndrome, sequencing and copy number analysis of BMPR1A should be considered in patients with (atrioventricular) septal defects, especially when associated with facial dysmorphism and anomalous growth.

Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair.

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.

Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation.

Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the large families MRX17 and MRX31. The minimal, commonly duplicated region contains three genes: RIBC1, HSD17B10, and HUWE1. RIBC1 could be excluded on the basis of its absence of expression in the brain and because it escapes X inactivation in females. For the other genes, expression array and quantitative PCR analysis in patient cell lines compared to controls showed a significant upregulation of HSD17B10 and HUWE1 as well as several important genes in their molecular pathways. Loss-of-function mutations of HSD17B10 have previously been associated with progressive neurological disease and XLMR. The E3 ubiquitin ligase HUWE1 has been implicated in TP53-associated regulation of the neuronal cell cycle. Here, we also report segregating sequence changes of highly conserved residues in HUWE1 in three XLMR families; these changes are possibly associated with the phenotype. Our findings demonstrate that an increased gene dosage of HSD17B10, HUWE1, or both contribute to the etiology of XLMR and suggest that point mutations in HUWE1 are associated with this disease too.

ZIC1 gene expression is controlled by DNA and histone methylation in mesenchymal proliferations.

RNA and protein analysis revealed the consistent upregulation of the neural transcription factors ZIC1 and ZIC4 in desmoid tumors and other fibroproliferative disorders. The 5' flanking region of the ZIC1 promoter was unmethylated in desmoid tumor fibroblasts, while a hypermethylated ZIC1 promoter was found in human and mouse cell lines not expressing the gene. In addition, expressing cells showed a H3K4me2 at the ZIC1 promoter, whereas non-expressing cells showed higher levels of H3K9me2 in the same region. To our knowledge, this is the first report describing ZIC1 expression in mesenchymal proliferations and a role for DNA methylation in the control of ZIC1 expression.

Detection of genomic copy number changes in patients with idiopathic mental retardation by high-resolution X-array-CGH: important role for increased gene dosage of XLMR genes.

A tiling X-chromosome-specific genomic array with a theoretical resolution of 80 kb was developed to screen patients with idiopathic mental retardation (MR) for submicroscopic copy number differences. Four patients with aberrations previously detected at lower resolution were first analyzed. This facilitated delineation of the location and extent of the aberration at high resolution and subsequently, more precise genotype-phenotype analyses. A cohort of 108 patients was screened, 57 of which were suspected of X-linked mental retardation (XLMR), 26 were probands of brother pairs, and 25 were sporadic cases. A total of 15 copy number changes in 14 patients (13%) were detected, which included two deletions and 13 duplications ranging from 0.1 to 2.7 Mb. The aberrations are associated with the phenotype in five patients (4.6%), based on the following criteria: de novo aberration; involvement of a known or candidate X-linked nonsyndromic(syndromic) MR (MRX(S)) gene; segregation with the disease in the family; absence in control individuals; and skewed X-inactivation in carrier females. These include deletions that contain the MRX(S) genes CDKL5, OPHN1, and CASK, and duplications harboring CDKL5, NXF5, MECP2, and GDI1. In addition, seven imbalances were apparent novel polymorphic regions because they do not fulfill the proposed criteria. Taken together, our data strongly suggest that not only deletions but also duplications on the X chromosome contribute to the phenotype more often than expected, supporting the increased gene dosage mechanism for deregulation of normal cognitive development.

Duplication of the MYB oncogene in T cell acute lymphoblastic leukemia.

We identified a duplication of the MYB oncogene in 8.4% of individuals with T cell acute lymphoblastic leukemia (T-ALL) and in five T-ALL cell lines. The duplication is associated with a threefold increase in MYB expression, and knockdown of MYB expression initiates T cell differentiation. Our results identify duplication of MYB as an oncogenic event and suggest that MYB could be a therapeutic target in human T-ALL.

Loss of SLC38A5 and FTSJ1 at Xp11.23 in three brothers with non-syndromic mental retardation due to a microdeletion in an unstable genomic region.

Using high resolution X chromosome array-CGH we identified an interstitial microdeletion at Xp11.23 in three brothers with moderate to severe mental retardation (MR) without dysmorphic features. The extent of the deletion was subsequently delineated to about 50 kb by regular PCR and included only the SLC38A5 and FTSJ1 genes. The loss of the FTSJ1 MR gene in males is expected to result in the observed phenotype but the contribution of the deletion of the solute carrier SLC38A5 gene is less clear. Their mother also carries the deletion and completely inactivates the aberrant X chromosome. Interestingly, the distal breakpoint is situated within a 200 kb SSX repeat region that appears to stimulate recombination since subtle copy number changes often occur at this location and it is frequently involved in translocations in tumours. Since this apparent SSX unstable structure is flanked proximally by FTSJ1 and PQBP1, subtle deletions or duplications at this location would be expected to cause MR, as in our family. So far, we have screened a cohort of 300 patients but did not find additional aberrations at the FTSJ1 locus indicating that the frequency is likely to be low.