X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

Phenotype and genotype in 101 males with X-linked creatine transporter deficiency.

BACKGROUND: Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and phenotype--genotype correlation has been lacking. METHODS: We performed a retrospective study of clinical, biochemical and molecular genetic data of 101 males with X-linked creatine transporter deficiency from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). RESULTS AND CONCLUSIONS: Most patients developed moderate to severe intellectual disability; mild intellectual disability was rare in adult patients. Speech language development was especially delayed but almost a third of the patients were able to speak in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine to creatinine ratio proved to be a reliable screening method besides MR spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. A third of patients had a de novo mutation in the SLC6A8 gene. Mothers with an affected son with a de novo mutation should be counselled about a recurrence risk in further pregnancies due to the possibility of low level somatic or germline mosaicism. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3' end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesise creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurones.

Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia.

The Wiskott-Aldrich syndrome protein (WASP; encoded by the gene WAS) and its homologs are important regulators of the actin cytoskeleton, mediating communication between Rho-family GTPases and the actin nucleation/crosslinking factor, the Arp2/3 complex. Many WAS mutations impair cytoskeletal control in hematopoietic tissues, resulting in functional and developmental defects that define the X-linked Wiskott-Aldrich syndrome (WAS) and the related X-linked thrombocytopenia (XLT). These diseases seem to result from reduced WASP signaling, often through decreased transcription or translation of the gene. Here we describe a new disease, X-linked severe congenital neutropenia (XLN), caused by a novel L270P mutation in the region of WAS encoding the conserved GTPase binding domain (GBD). In vitro, the mutant protein is constitutively activated through disruption of an autoinhibitory domain in the wild-type protein, indicating that loss of WASP autoinhibition is a key event in XLN. Our findings highlight the importance of precise regulation of WASP in hematopoietic development and function, as impairment versus enhancement of its activity give rise to distinct spectra of cellular defects and clinical phenotypes.

A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation.

X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%-1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including beta-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.

A new member of the IL-1 receptor family highly expressed in hippocampus and involved in X-linked mental retardation.

We demonstrate here the importance of interleukin signalling pathways in cognitive function and the normal physiology of the CNS. Thorough investigation of an MRX critical region in Xp22.1-21.3 enabled us to identify a new gene expressed in brain that is responsible for a non-specific form of X-linked mental retardation. This gene encodes a 696 amino acid protein that has homology to IL-1 receptor accessory proteins. Non-overlapping deletions and a nonsense mutation in this gene were identified in patients with cognitive impairment only. Its high level of expression in post-natal brain structures involved in the hippocampal memory system suggests a specialized role for this new gene in the physiological processes underlying memory and learning abilities.

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.

Chromosomal copy number changes in patients with non-syndromic X linked mental retardation detected by array CGH.

Several studies have shown that array based comparative genomic hybridisation (CGH) is a powerful tool for the detection of copy number changes in the genome of individuals with a congenital disorder. In this study, 40 patients with non-specific X linked mental retardation were analysed with full coverage, X chromosomal, bacterial artificial chromosome arrays. Copy number changes were validated by multiplex ligation dependent probe amplification as a fast method to detect duplications and deletions in patient and control DNA. This approach has the capacity to detect copy number changes as small as 100 kb. We identified three causative duplications: one family with a 7 Mb duplication in Xp22.2 and two families with a 500 kb duplication in Xq28 encompassing the MECP2 gene. In addition, we detected four regions with copy number changes that were frequently identified in our group of patients and therefore most likely represent genomic polymorphisms. These results confirm the power of array CGH as a diagnostic tool, but also emphasise the necessity to perform proper validation experiments by an independent technique.

[What if the sperm donor has a hereditary disease? Informed consent needed for sharing medical information]. / Wat als de zaaddonor een erfelijke ziekte heeft?

There are no regulations in the Netherlands regarding the exchange of important genetic information that has become available after the birth of a child conceived with donor gametes. This may lead to difficult situations such as when the gamete donor is found to suffer from a genetic cancer-predisposition disorder. Genetic information about the donor that becomes available later may be of great importance to donor offspring. Genetic information uncovered in the donor child may likewise be of importance to legal offspring of the gamete donor. We propose an informed-consent procedure for both donors and recipients to take better care of this issue.

A third MRX family (MRX68) is the result of mutation in the long chain fatty acid-CoA ligase 4 (FACL4) gene: proposal of a rapid enzymatic assay for screening mentally retarded patients.

BACKGROUND: The gene encoding fatty acid CoA ligase 4 (FACL4) is mutated in families with non-specific X linked mental retardation (MRX) and is responsible for cognitive impairment in the contiguous gene syndrome ATS-MR (Alport syndrome and mental retardation), mapped to Xq22.3. This finding makes this gene a good candidate for other mental retardation disorders mapping in this region. METHODS: We have screened the FACL4 gene in eight families, two MRX and six syndromic X linked mental retardation (MRXS), mapping in a large interval encompassing Xq22.3. RESULTS: We have found a missense mutation in MRX68. The mutation (c.1001C>T in the brain isoform) cosegregates with the disease and changes a highly conserved proline into a leucine (p.P375L) in the first luciferase domain, which markedly reduces the enzymatic activity. Furthermore, all heterozygous females showed completely skewed X inactivation in blood leucocytes, as happens in all reported females with other FACL4 point mutations or deletions. CONCLUSIONS: Since the FACL4 gene is highly expressed in brain, where it encodes a brain specific isoform, and is located in hippocampal and cerebellar neurones, a role for this gene in cognitive processes can be expected. Here we report the third MRX family with a FACL4 mutation and describe the development of a rapid enzymatic assay on peripheral blood that we propose as a sensitive, robust, and efficient diagnostic tool in mentally retarded males.

De novo 7q36 deletion: breakpoint analysis and types of holoprosencephaly.

We report on a de novo 7q36 deletion in a 3-month-old girl with manifestations of the 7q terminal deletion syndrome. Only minimal findings of holoprosencephaly (HPE) were present since only a partial corpus callosum hypoplasia was seen on a magnetic resonance imaging scan of the brain. Extensive fluorescence in situ hybridization analysis showed that the HPE3 critical gene region, inclusive Sonic hedgehog (SHH), En2 (HOX1), and HTR5A, was deleted. A review of 33 other patients with a de novo terminal 7q deletion and the different types of HPE manifestations within these patients will be presented.

Variable expression of phenotype in offspring with partial monosomy 7q and partial trisomy 8p in a family with a rcp (7;8)(134;p12) translocation.

We report a female fetus with partial 7q monosomy and partial 8p trisomy, as the unbalanced product of a familial balanced reciprocal translocation; rcp t(7;8)(134:p12)mat. Among pregnancies from translocation carriers in this family, there has been a high incidence of first trimester miscarriages. Three unbalanced offsprings with a partial 7q monosomy and partial 8p trisomy were diagnosed after prenatal investigations. One female fetus with this unbalanced karyotype has previously been reported. She had a multiple congenital anomalies (MCA) syndrome. In contrast, the present female fetus with the same abnormal karyotype shows only mild facial dysmorfism.

Partial trisomy and partial monosomy of the distal long arm of chromosome 4: patient report and literature review.

We report the first patient with a partial trisomy and a partial monosomy of the long arm of chromosome 4: 46,XY, inv dup(4)(pter-->q32::q32-->q26), del(4)(q32-->qter). The boy died from a complex cardiac defect (monoventricle, monoatrium and truncus arteriosus) in combination with a diaphragmatic hernia. In addition he had preaxial polydactyly of the right hand. We compare the clinical features with data from the literature. The phenotype of the patient mainly resembles that in patients with a terminal deletion 4q32.

Holoprosencephaly: the Maastricht experience.

Holoprosencephaly (HPE) is a developmental field defect with impaired cleavage of the embryonic forebrain as the cardinal feature. The prevalence is about 1 in 11.000-20.000 in live births and 1 in 250 during embryogenesis. In most cases, craniofacial abnormalities are associated and reflect in 80% of cases the degree of severity. The severity is of marked variability and ranges from cyclopia to minimal craniofacial dysmorphism, such as mild microcephaly with a single central incisor. The etiology of HPE is very heterogeneous and comprises environmental factors (e.g. maternal diabetes) and genetic causes. Approximately 50% of HPE cases are associated with a cytogenetic abnormality (the most common of which is trisomy 13) or a monogenic syndrome. Based on recurrent cytogenetic abnormalities, there are at least 12 genetic loci that likely contain genes implicated in the pathogenesis of HPE. Currently, four human HPE genes are known: SHH at 7q36, ZIC2 at 13q32, SIX3 at 2p21 and TGIF at 18p11.3. Over the past 13 years, 16 patients with HPE have been observed at the Department of Clinical Genetics at Maastricht. Some of them are briefly presented in order to emphasize the spectral nature of HPE and the etiological heterogeneity. One patient appeared to have a partial 18p deletion due to a maternal cryptic translocation t(1:18) and, in addition, a SHH mutation. The mildest affected patient presented with microcephaly and a single maxillary incisor; she had a submicroscopic 7q deletion. Finally, we propose a protocol of etiological work-up of HPE cases.

Strong variable clinical presentation in 3 patients with 7q terminal deletion.

We report 3 patients with a 7q terminal deletion. The first, a 7 weeks old female, with a de novo 7q36-->qter deletion, was microcephalic and had a partial hypoplasia of the corpus callosum on the MRI-scan of the brain. The second, a 3 months old male, showed microcephaly, disproportionate growth retardation, truncal obesity and facial dysmorfism giving the clinical impression of a "microcephalic primordial dwarfism (osteodysplastic type)". At the age of 6 months he had developed a single maxillary central incisor suggesting a minimal form of holoprosencephaly (HPE). Additional FISH-studies showed a 7q36.1-->qter deletion, as the unbalanced product of a t(5;7)(q35.2;q36.1)pat. The de novo 7q36-->qter deletion in the third patient, a 5 years old female, was associated with borderline intelligence, mild microcephaly, small midface, choanal narrowing and a single maxillary central incisor as a minimal form of HPE. CT- and MRI-scan of the brain were normal. In these 3 patients extensive FISH analysis was performed to investigate the possible involvement of the HPE gene region on chromosome 7q36. The target gene for HPE, the Sonic hedgehog gene (SHH) as well as several other genes important for normal brain development (En2;HOX1,HTR5A) were found to be deleted in all three patients. Our findings stress the importance of 7q36 microdeletion studies in patients with even minimal signs of HPE, as relative microcephaly with small midface (choanal narrowing), agenesis/hypoplasia of the corpus callosum/septum pellucidum, thalamic fusion or a single maxillary central incisor.

A young female with asymmetric manifestations of larsen syndrome: another example of unilateral somatic cell-line mosaicism.

Larsen syndrome is characterized by multiple congenital joint dislocations, typical skeletal defects and facial dysmorphism. In this article, we present a female patient with asymmetric Larsen syndrome. We hypothesise that the asymmetric distribution of clinical features in our patient is likely caused by post-zygotic somatic cell-line mosaicism of a dominant gene mutation.

Noninvasive detection of fetal trisomy 21: systematic review and report of quality and outcomes of diagnostic accuracy studies performed between 1997 and 2012.

BACKGROUND: Research on noninvasive prenatal testing (NIPT) of fetal trisomy 21 is developing fast. Commercial tests have become available. To provide an up-to-date overview of NIPT of trisomy 21, an evaluation of the methodological quality and outcomes of diagnostic accuracy studies was made. METHODS: We undertook a systematic review of the literature published between 1997 and 2012 after searching PubMed, using MeSH terms 'RNA', 'DNA' and 'Down Syndrome' in combination with 'cell-free fetal (cff) RNA', 'cffDNA', 'trisomy 21' and 'noninvasive prenatal diagnosis' and searching reference lists of reported literature. From 79 abstracts, 16 studies were included as they evaluated the diagnostic accuracy of a molecular technique for NIPT of trisomy 21, and the test sensitivity and specificity were reported. Meta-analysis could not be performed due to the use of six different molecular techniques and different cutoff points. Diagnostic parameters were derived or calculated, and possible bias and applicability were evaluated utilizing the revised tool for Quality Assessment of Diagnostic Accuracy (QUADAS-2). RESULTS: Seven of the included studies were recently published in large cohort studies that examined massively parallel sequencing (MPS), with or without pre-selection of chromosomes, and reported sensitivities between 98.58% [95% confidence interval (CI) 95.9-99.5%] and 100% (95% CI 96-100%) and specificities between 97.95% (95% CI 94.1-99.3%) and 100% (95% CI 99.1-100%). None of these seven large studies had an overall low risk of bias and low concerns regarding applicability. MPS with or without pre-selection of chromosomes exhibits an excellent negative predictive value (100%) in conditions with disease odds from 1:1500 to 1:200. However, positive predictive values were lower, even in high-risk pregnancies (19.7-100%). The other nine cohort studies were too small to give precise estimates (number of trisomy 21 cases: ≤25) and were not included in the discussion. CONCLUSIONS: NIPT of trisomy 21 by MPS with or without pre-selection of chromosomes is promising and likely to replace the prenatal serum screening test that is currently combined with nuchal translucency measurement in the first trimester of pregnancy. Before NIPT can be introduced as a screening test in a social insurance health-care system, more evidence is needed from large prospective diagnostic accuracy studies in first trimester pregnancies. Moreover, we believe further assessment, of whether NIPT can be provided in a cost-effective, timely and equitable manner for every pregnant woman, is required.

Adducted thumbs: a clinical clue to genetic diagnosis.

Adducted thumbs are an uncommon congenital malformation. It can be an important clinical clue in genetic syndromes, e.g. the L1 syndrome. A retrospective survey was performed including patients with adducted thumbs referred to the Department of Clinical Genetics between 1985 and 2011 by perinatologists, (child) neurologists or paediatricians, in order to evaluate current knowledge on the genetic etiology of adducted thumbs. Twenty-five patients were included in this survey. Additional features were observed in 88% (22/25). In 25% (4/16) of the patients with adducted thumbs and congenital hydrocephalus L1CAM gene mutations were identified. One patient had a mosaic 5p13 duplication. Recommendations are made concerning the evaluation and genetic workup of patients with adducted thumbs.

Congenital hydrocephalus in clinical practice: a genetic diagnostic approach.

Congenital hydrocephalus is a common and often disabling disorder. The etiology is very heterogeneous. Little is known about the genetic causes of congenital hydrocephalus. A retrospective survey was performed including patients with primary congenital hydrocephalus referred to the Department of Clinical Genetics between 1985 and 2010 by perinatologists, (child) neurologists or pediatricians. Patients with hydrocephalus secondary to other pathology were excluded from this survey. We classified patients with primary congenital hydrocephalus into two main groups: non-syndromic hydrocephalus (NSH) and syndromic hydrocephalus (SH). Seventy-five individuals met the inclusion criteria, comprising 36% (27/75) NSH and 64% (48/75) SH. In 11% (8/75) hydrocephalus was familial. The cause of hydrocephalus was unknown in 81% (61/75), including all patients with NSH. The male-female ratio in this subgroup was 2.6:1, indicating an X-linked factor other than the L1CAM gene. In the group of SH patients, 29% (14/48) had a known cause of hydrocephalus including chromosomal abnormalities, L1 syndrome, Marden-Walker syndrome, Walker-Warburg syndrome and hemifacial microsomia. We performed this survey in order to evaluate current knowledge on the genetic etiology of primary congenital hydrocephalus and to identify new candidate genes or regulatory pathways for congenital hydrocephalus. Recommendations were made concerning the evaluation and genetic workup of patients with primary congenital hydrocephalus. We conclude that further molecular and functional analysis is needed to identify new genetic forms of congenital hydrocephalus.