A.P.M. de Brouwer

Disruption of the gene Euchromatin Histone Methyl Transferase1 (Eu-HMTase1) is associated with the 9q34 subtelomeric deletion syndrome

Background: A new syndrome has been recognised following thorough analysis of patients with a terminal submicroscopic subtelomeric deletion of chromosome 9q. These have in common severe mental retardation, hypotonia, brachycephaly, flat face with hypertelorism, synophrys, anteverted nares, thickened lower lip, carp mouth with macroglossia, and conotruncal heart defects. The minimum critical region responsible for this 9q subtelomeric deletion syndrome (9q−) is approximately 1.2 Mb and encompasses at least 14 genes. Objective: To characterise the breakpoints of a de novo balanced translocation t(X;9)(p11.23;q34.3) in a mentally retarded female patient with clinical features similar to the 9q− syndrome. Results: Sequence analysis of the break points showed that the translocation was fully balanced and only one gene on chromosome 9 was disrupted—Euchromatin Histone Methyl Transferase1 (Eu-HMTase1)—encoding a histone H3 lysine 9 methyltransferase (H3-K9 HMTase). This indicates that haploinsufficiency of Eu-HMTase1 is responsible for the 9q submicroscopic subtelomeric deletion syndrome. This observation was further supported by the spatio-temporal expression of the gene. Using tissue in situ hybridisation studies in mouse embryos and adult brain, Eu-HMTase1 was shown to be expressed in the developing nervous system and in specific peripheral tissues. While expression is selectively downregulated in adult brain, substantial expression is retained in the olfactory bulb, anterior/ventral lateral ventricular wall, and hippocampus and weakly in the piriform cortex. Conclusions: The expression pattern of this gene suggests a role in the CNS development and function, which is in line with the severe mental retardation and behaviour problems in patients who lack one copy of the gene.

Chromosome 1p21.3 microdeletions comprising DPYD and MIR137 are associated with intellectual disability

Background MicroRNAs (miRNAs) are non-coding gene transcripts involved in post-transcriptional regulation of genes. Recent studies identified miRNAs as important regulators of learning and memory in model organisms. So far, no mutations in specific miRNA genes have been associated with impaired cognitive functions. Methods and results In three sibs and two unrelated patients with intellectual disability (ID), overlapping 1p21.3 deletions were detected by genome-wide array analysis. The shortest region of overlap included dihydropyrimidine dehydrogenase (DPYD) and microRNA 137 (MIR137). DPYD is involved in autosomal recessive dihydropyrimidine dehydrogenase deficiency. Hemizygous DPYD deletions were previously suggested to contribute to a phenotype with autism spectrum disorder and speech delay. Interestingly, the mature microRNA transcript microRNA-137 (miR-137) was recently shown to be involved in modulating neurogenesis in adult murine neuronal stem cells. Therefore, this study investigated the possible involvement of MIR137 in the 1p21.3-deletion phenotype. The patients displayed a significantly decreased expression of both precursor and mature miR-137 levels, as well as significantly increased expression of the validated downstream targets microphthalmia-associated transcription factor (MITF) and Enhancer of Zeste, Drosophila, Homologue 2 (EZH2), and the newly identified target Kruppel-like factor 4 (KLF4). The study also demonstrated significant enrichment of miR-137 at the synapses of cortical and hippocampal neurons, suggesting a role of miR-137 in regulating local synaptic protein synthesis machinery. Conclusions This study showed that dosage effects of MIR137 are associated with 1p21.3 microdeletions and may therefore contribute to the ID phenotype in patients with deletions harbouring this miRNA. A local effect at the synapse might be responsible.

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.

Clinical significance of de novo and inherited copy-number variation.

Copy‐number variations (CNVs) are a common cause of intellectual disability and/or multiple congenital anomalies (ID/MCA). However, the clinical interpretation of CNVs remains challenging, especially for inherited CNVs. Well‐phenotyped patients (5,531) with ID/MCA were screened for rare CNVs using a 250K single‐nucleotide polymorphism array platform in order to improve the understanding of the contribution of CNVs to a patients phenotype. We detected 1,663 rare CNVs in 1,388 patients (25.1%; range 0–5 per patient) of which 437 occurred de novo and 638 were inherited. The detected CNVs were analyzed for various characteristics, gene content, and genotype–phenotype correlations. Patients with severe phenotypes, including organ malformations, had more de novo CNVs (P < 0.001), whereas patient groups with milder phenotypes, such as facial dysmorphisms, were enriched for both de novo and inherited CNVs (P < 0.001), indicating that not only de novo but also inherited CNVs can be associated with a clinically relevant phenotype. Moreover, patients with multiple CNVs presented with a more severe phenotype than patients with a single CNV (P < 0.001), pointing to a combinatorial effect of the additional CNVs. In addition, we identified 20 de novo single‐gene CNVs that directly indicate novel genes for ID/MCA, including ZFHX4, ANKH, DLG2, MPP7, CEP89, TRIO, ASTN2, and PIK3C3.

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.

Involvement of the kinesin family members KIF4A and KIF5C in intellectual disability and synaptic function

Introduction Kinesin superfamily (KIF) genes encode motor proteins that have fundamental roles in brain functioning, development, survival and plasticity by regulating the transport of cargo along microtubules within axons, dendrites and synapses. Mouse knockout studies support these important functions in the nervous system. The role of KIF genes in intellectual disability (ID) has so far received limited attention, although previous studies have suggested that many ID genes impinge on synaptic function. Methods By applying next-generation sequencing (NGS) in ID patients, we identified likely pathogenic mutations in KIF4A and KIF5C. To further confirm the pathogenicity of these mutations, we performed functional studies at the level of synaptic function in primary rat hippocampal neurons. Results and conclusions Four males from a single family with a disruptive mutation in the X-linked KIF4A (c.1489-8_1490delins10; p.?- exon skipping) showed mild to moderate ID and epilepsy. A female patient with a de novo missense mutation in KIF5C (c.11465A>C; p.(Glu237Lys)) presented with severe ID, epilepsy, microcephaly and cortical malformation. Knock-down of Kif4a in rat primary hippocampal neurons altered the balance between excitatory and inhibitory synaptic transmission, whereas the mutation in Kif5c affected its protein function at excitatory synapses. Our results suggest that mutations in KIF4A and KIF5C cause ID by tipping the balance between excitatory and inhibitory synaptic excitability.

Variable clinical features in patients with CDH23 mutations (USH1D-DFNB12).

Objective: To describe the findings of audiovestibular and ophthalmologic examinations in four families with mutations in the CDH23 gene. Study Design: Family study. Setting: Tertiary referral center. Patients: Four DFNB12 patients from a large consanguineous Dutch family and six patients from three different Usher syndrome Type ID families were examined. All were identified by at least one pathogenic mutation in the CDH23 gene. Methods: Audiovestibular examinations consisted of standard pure-tone audiometry, vestibulo-ocular reflex, optokinetic nystagmus, and in some cases the cervico-ocular reflex. Linear regression analysis was used to evaluate progression of hearing impairment, and the degree of hearing impairment of DFNB12 was compared with that found for USH1D. Ophthalmologic examinations consisted of best-corrected visual acuity, Goldmann perimetry, slit-lamp examinations, color vision testing, dark adaptation, electroretinography, electro-oculography, funduscopy and photography of the retina, and sometimes fluorescein angiography. Results: The USH1D patients had significantly worse hearing impairment than the DFNB12 patients. The DFNB12 patients, identified by missense mutations in CDH23, had normal retinal and vestibular function. All USH1D patients had splice-site mutations in CDH23 and a typical Usher syndrome Type I phenotype. One DFNB12 patient had slightly abnormal yellowish flecks in the posterior poles of both eyes. Conclusion: Recessive missense mutations in CDH23 lead to a milder phenotype (DFNB12) than splice-site mutations (USH1D); however, abnormal bilateral flecks, suggestive for lipofuscin accumulation, can be observed in DFNB12 patients.

A novel locus for autosomal dominant non-syndromic hearing loss, DFNA31, maps to chromosome 6p21.3.

Background:Non-syndromic hearing loss is the most genetically heterogeneous trait known in humans. To date, 51 loci for autosomal dominant non-syndromic sensorineural hearing loss (NSSHL) have been identified by linkage analysis. Objective:To investigate the genes involved in a Dutch family with NSSHL. Methods:Linkage analysis in a large Dutch pedigree with progressive bilateral loss of the mid and high frequencies, in which a novel dominant locus for postlingual NSSHL (DFNA31) has been identified. Results:DFNA31 was found to be located in a 7.5 cM region of chromosome 6p21.3 between D6S276 (telomeric) and D6S273 (centromeric), with a maximum two point LOD score of 5.99 for D6S1624. DNA sequencing of coding regions and exon/intron boundaries of two candidate genes (POU5F1, GABBR1) in this interval did not reveal disease causing mutations. Conclusions:Haplotype analysis indicated that the genetic defect in this family does not overlap the DFNA13 and DFNA21 regions that are also located on 6p. Identification of the disease gene will be of major importance in understanding the pathophysiology of hearing impairment.

Refining the critical region of the novel 19q13.11 microdeletion syndrome to 750 Kb

With great interest we read the article of Malan et al , who reported on a novel clinically recognisable 19q13.11 microdeletion syndrome.1 Here we report on a fifth patient with an interstitial deletion overlapping the 19q13.11 region and compare our findings with those described by Malan et al . The proband was born after 37 weeks of gestation as one of dizygotic twins with a birth weight of 1620 g (−3.5 SD). His twin sister had a normal birth weight of 2290 g (p25). They were the first children of healthy non-consanguineous parents. Pregnancy was complicated by intrauterine growth retardation. At birth the proband had cutis aplasia over the posterior occiput, hypospadias, abnormal positioning of the feet, and a third nipple on the left side of the chest (the latter was also present in the father) whereas no dysmorphisms were reported in his sister. During the first years of life, feeding difficulties, failure to thrive, and psychomotor delay were noted: he sat independently at 2 years of age, started crawling at the same age, and started walking at the age of 3 years and 10 months. Because of fatigue, he was able to walk short distances only, and walked with a stiff gait. He suffered from recurrent airway infections. At the age of 4 years and 10 months he could not speak, but was able to communicate with pictograms. He had hypermetropia (+3D/+4D) similar to his sister. At …

A 3-base pair deletion, c.9711_9713del, in DMD results in intellectual disability without muscular dystrophy

We have identified a deletion of 3 base pairs in the dystrophin gene (DMD), c.9711_9713del, in a family with nonspecific X-linked intellectual disability (ID) by sequencing of the exons of 86 known X-linked ID genes. This in-frame deletion results in the deletion of a single-amino-acid residue, Leu3238, in the brain-specific isoform Dp71 of dystrophin. Linkage analysis supported causality as the mutation was present in the 7.6 cM linkage interval on Xp22.11–Xp21.1 with a maximum positive LOD score of 2.41 (MRX85 locus). Molecular modeling predicts that the p.(Leu3238del) deletion results in the destabilization of the C-terminal domain of dystrophin and hence reduces the ability to interact with β-dystroglycan. Correspondingly, Dp71 protein levels in lymphoblastoid cells from the index patient are 6.7-fold lower than those in control cell lines (P=0.08). Subsequent determination of the creatine kinase levels in blood of the index patient showed a mild but significant elevation in serum creatine kinase, which is in line with impaired dystrophin function. In conclusion, we have identified the first DMD mutation in Dp71 that results in ID without muscular dystrophy.