Guy Froyen

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.

Mild Wolf-Hirschhorn syndrome: micro-array CGH analysis of atypical 4p16.3 deletions enables refinement of the genotype-phenotype map

Wolf-Hirschhorn syndrome is a multiple malformation syndrome with distinct abnormal craniofacial features, prenatal onset growth retardation, failure to thrive, microcephaly, usually severe mental retardation, seizures, and congenital heart malformations. Large variations are observed in phenotypic expression of these features, with mental retardation ranging from severe to mild. There is a one third mortality in the first two years of life. Most patients with Wolf-Hirschhorn syndrome carry 4p terminal deletions. However, the size of these deletions is variable and several phenotypic features have been tentatively mapped within the 4pter region.1–4 Further fine mapping of the different phenotypic features will ultimately lead to a functional understanding of the genes that cause these abnormal phenotypes. The minimal ‘Wolf-Hirschhorn syndrome’ phenotype was defined as the typical facial appearance, congenital hypotonia, mental retardation, growth delay, and seizures.2,4 The Wolf-Hirschhorn syndrome critical region was originally confined to a region of 165 kb and nine transcripts within this region were described.5 A patient with a small intrachromosomal 4p deletion and a partial Wolf-Hirschhorn syndrome phenotype further refined the critical region (WHSCR1).6 Two genes, the Wolf-Hirschhorn Syndrome Candidate genes 1 (WHSC1) and 2 (WHSC2), are located in the region. The expression pattern of WHSC1 colocalises spatially and temporarily with the major Wolf-Hirschhorn syndrome malformations and the gene is homologous with a Drosophila dysmorphology gene.7 WHSC2 is a nuclear protein with a helix-loop-helix motif that is ubiquitously expressed throughout development.8,9 The identification of a Wolf-Hirschhorn syndrome patient with a terminal 1.9 Mb deletion not including this Wolf-Hirschhorn syndrome critical region led Zollino et al4 to postulate a novel critical region distal to the previously defined critical region, which was termed the Wolf-Hirschhorn critical region 2 (WHSCR2). The distal boundary of this region is located within the WHSCR1 and at …

NXF5, a novel member of the nuclear RNA export factor family, is lost in a male patient with a syndromic form of mental retardation

BACKGROUND Although X-linked mental retardation (XLMR) affects 2%-3% of the human population, little is known about the underlying molecular mechanisms. Recent interest in this topic led to the identification of several genes for which mutations result in the disturbance of cognitive development. RESULTS We identified a novel gene that is interrupted by an inv(X)(p21.1;q22) in a male patient with a syndromic form of mental retardation. Molecular analysis of both breakpoint regions did not reveal an interrupted gene on Xp, but identified a novel nuclear RNA export factor (NXF) gene cluster, Xcen-NXF5-NXF2-NXF4-NXF3-Xqter, in which NXF5 is split by the breakpoint, leading to its functional nullisomy. The predicted NXF5 protein shows high similarity with the central part of the presumed mRNA nuclear export factor TAP/NXF1. Functional analysis of NXF5 demonstrates binding to RNA as well as to the RNA nuclear export-associated protein p15/NXT. In contrast to TAP/NXF1, overexpression studies localized NXF5 in the form of granules in the cell body and neurites of mature hippocampal neurons, suggesting a role in mRNA transport. The two newly identified mouse nxf homologs, nxf-a and nxf-b, which also map on X, show highest mRNA levels in the brain. CONCLUSIONS A novel member of the nuclear RNA export factor family is absent in a male patient with a syndromic form of mental retardation. Although we did not find direct evidence for the involvement of NXF5 in MR, the gene could be involved in development, possibly through a process in mRNA metabolism in neurons.

A noncoding, regulatory mutation implicates HCFC1 in nonsyndromic intellectual disability

The discovery of mutations causing human disease has so far been biased toward protein-coding regions. Having excluded all annotated coding regions, we performed targeted massively parallel resequencing of the nonrepetitive genomic linkage interval at Xq28 of family MRX3. We identified in the binding site of transcription factor YY1 a regulatory mutation that leads to overexpression of the chromatin-associated transcriptional regulator HCFC1. When tested on embryonic murine neural stem cells and embryonic hippocampal neurons, HCFC1 overexpression led to a significant increase of the production of astrocytes and a considerable reduction in neurite growth. Two other nonsynonymous, potentially deleterious changes have been identified by X-exome sequencing in individuals with intellectual disability, implicating HCFC1 in normal brain function.

Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing

Massive parallel sequencing has revolutionized the search for pathogenic variants in the human genome, but for routine diagnosis, re-sequencing of the complete human genome in a large cohort of patients is still far too expensive. Recently, novel genome partitioning methods have been developed that allow to target re-sequencing to specific genomic compartments, but practical experience with these methods is still limited. In this study, we have combined a novel droplet-based multiplex PCR method and next generation sequencing to screen patients with X-linked mental retardation (XLMR) for mutations in 86 previously identified XLMR genes. In total, affected males from 24 large XLMR families were analyzed, including three in whom the mutations were already known. Amplicons corresponding to functionally relevant regions of these genes were sequenced on an Illumina/Solexa Genome Analyzer II platform. Highly specific and uniform enrichment was achieved: on average, 67.9% unambiguously mapped reads were derived from amplicons, and for 88.5% of the targeted bases, the sequencing depth was sufficient to reliably detect variations. Potentially disease-causing sequence variants were identified in 10 out of 24 patients, including the three mutations that were already known, and all of these could be confirmed by Sanger sequencing. The robust performance of this approach demonstrates the general utility of droplet-based multiplex PCR for parallel mutation screening in hundreds of genes, which is a prerequisite for the diagnosis of mental retardation and other disorders that may be due to defects of a wide variety of genes.

Large-scale analysis of tandem repeat variability in the human genome

Tandem repeats are short DNA sequences that are repeated head-to-tail with a propensity to be variable. They constitute a significant proportion of the human genome, also occurring within coding and regulatory regions. Variation in these repeats can alter the function and/or expression of genes allowing organisms to swiftly adapt to novel environments. Importantly, some repeat expansions have also been linked to certain neurodegenerative diseases. Therefore, accurate sequencing of tandem repeats could contribute to our understanding of common phenotypic variability and might uncover missing genetic factors in idiopathic clinical conditions. However, despite long-standing evidence for the functional role of repeats, they are largely ignored because of technical limitations in sequencing, mapping and typing. Here, we report on a novel capture technique and data filtering protocol that allowed simultaneous sequencing of thousands of tandem repeats in the human genomes of a three generation family using GS-FLX-plus Titanium technology. Our results demonstrated that up to 7.6% of tandem repeats in this family (4% in coding sequences) differ from the reference sequence, and identified a de novo variation in the family tree. The method opens new routes to look at this underappreciated type of genetic variability, including the identification of novel disease-related repeats.

Early frameshift mutation in PIGA identified in a large XLID family without neonatal lethality.

The phosphatidylinositol glycan class A (PIGA) protein is a member of the glycosylphosphatidylinositol anchor pathway. Germline mutations in PIGA located at Xp22.2 are thought to be lethal in males. However, a nonsense mutation in the last coding exon was recently described in two brothers with multiple congenital anomalies‐hypotonia‐seizures syndrome 2 (MCAHS2) who survived through birth likely because of the hypomorphic nature of the truncated protein, but died in their first weeks of life. Here, we report on a frameshift mutation early in the PIGA cDNA (c.76dupT; p.Y26Lfs*3) that cosegregates with the disease in a large family diagnosed with a severe syndromic form of X‐linked intellectual disability. Unexpectedly, CD59 surface expression suggested the production of a shorter PIGA protein with residual functionality. We provide evidence that the second methionine at position 37 may be used for the translation of a 36 amino acids shorter PIGA. Complementation assays confirmed that this shorter PIGA cDNA was able to partially rescue the surface expression of CD59 in a PIGA‐null cell line. Taken together, our data strongly suggest that the early frameshift mutation in PIGA produces a truncated hypomorph, which is sufficient to rescue the lethality in males but not the MCAHS2‐like phenotype.

Increased dosage of RAB39B affects neuronal development and could explain the cognitive impairment in male patients with distal Xq28 copy number gains.

Copy number gains at Xq28 are a frequent cause of X‐linked intellectual disability (XLID). Here, we report on a recurrent 0.5 Mb tandem copy number gain at distal Xq28 not including MECP2, in four male patients with nonsyndromic mild ID and behavioral problems. The genomic region is duplicated in two families and triplicated in a third reflected by more distinctive clinical features. The X‐inactivation patterns in carrier females correspond well with their clinical symptoms. Our mapping data confirm that this recurrent gain is likely mediated by nonallelic homologous recombination between two directly oriented Int22h repeats. The affected region harbors eight genes of which RAB39B encoding a small GTPase, was the prime candidate since loss‐of‐function mutations had been linked to ID. RAB39B is expressed at stable levels in lymphocytes from control individuals, suggesting a tight regulation. mRNA levels in our patients were almost two‐fold increased. Overexpression of Rab39b in mouse primary hippocampal neurons demonstrated a significant decrease in neuronal branching as well as in the number of synapses when compared with the control neurons. Taken together, we provide evidence that the increased dosage of RAB39B causes a disturbed neuronal development leading to cognitive impairment in patients with this recurrent copy number gain.

Partial duplications of the ATRX gene cause the ATR-X syndrome

ATR-X syndrome is a rare syndromic X-linked mental retardation disorder. We report that some of the patients suspected of ATR-X carry large intragenic duplications in the ATRX gene, leading to an absence of ATRX mRNA and of the protein. These findings underscore the need for including quantitative analyses to mutation analysis of the ATRX gene.

Identification of Intellectual Disability Genes in Female Patients with a Skewed X-Inactivation Pattern.

Intellectual disability (ID) is a heterogeneous disorder with an unknown molecular etiology in many cases. Previously, X‐linked ID (XLID) studies focused on males because of the hemizygous state of their X chromosome. Carrier females are generally unaffected because of the presence of a second normal allele, or inactivation of the mutant X chromosome in most of their cells (skewing). However, in female ID patients, we hypothesized that the presence of skewing of X‐inactivation would be an indicator for an X chromosomal ID cause. We analyzed the X‐inactivation patterns of 288 females with ID, and found that 22 (7.6%) had extreme skewing (>90%), which is significantly higher than observed in the general population (3.6%; P = 0.029). Whole‐exome sequencing of 19 females with extreme skewing revealed causal variants in six females in the XLID genes DDX3X, NHS, WDR45, MECP2, and SMC1A. Interestingly, variants in genes escaping X‐inactivation presumably cause both XLID and skewing of X‐inactivation in three of these patients. Moreover, variants likely accounting for skewing only were detected in MED12, HDAC8, and TAF9B. All tested candidate causative variants were de novo events. Hence, extreme skewing is a good indicator for the presence of X‐linked variants in female patients.