Christiane Zweier

Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study

BACKGROUND The genetic cause of intellectual disability in most patients is unclear because of the absence of morphological clues, information about the position of such genes, and suitable screening methods. Our aim was to identify de-novo variants in individuals with sporadic non-syndromic intellectual disability. METHODS In this study, we enrolled children with intellectual disability and their parents from ten centres in Germany and Switzerland. We compared exome sequences between patients and their parents to identify de-novo variants. 20 children and their parents from the KORA Augsburg Diabetes Family Study were investigated as controls. FINDINGS We enrolled 51 participants from the German Mental Retardation Network. 45 (88%) participants in the case group and 14 (70%) in the control group had de-novo variants. We identified 87 de-novo variants in the case group, with an exomic mutation rate of 1·71 per individual per generation. In the control group we identified 24 de-novo variants, which is 1·2 events per individual per generation. More participants in the case group had loss-of-function variants than in the control group (20/51 vs 2/20; p=0·022), suggesting their contribution to disease development. 16 patients carried de-novo variants in known intellectual disability genes with three recurrently mutated genes (STXBP1, SYNGAP1, and SCN2A). We deemed at least six loss-of-function mutations in six novel genes to be disease causing. We also identified several missense alterations with potential pathogenicity. INTERPRETATION After exclusion of copy-number variants, de-novo point mutations and small indels are associated with severe, sporadic non-syndromic intellectual disability, accounting for 45-55% of patients with high locus heterogeneity. Autosomal recessive inheritance seems to contribute little in the outbred population investigated. The large number of de-novo variants in known intellectual disability genes is only partially attributable to known non-specific phenotypes. Several patients did not meet the expected syndromic manifestation, suggesting a strong bias in present clinical syndrome descriptions. FUNDING German Ministry of Education and Research, European Commission 7th Framework Program, and Swiss National Science Foundation.

Transcription Factor E2-2 Is an Essential and Specific Regulator of Plasmacytoid Dendritic Cell Development

Plasmacytoid dendritic cells (PDCs) represent a unique immune cell type specialized in type I interferon (IFN) secretion in response to viral nucleic acids. The molecular control of PDC lineage specification has been poorly understood. We report that basic helix-loop-helix transcription factor (E protein) E2-2/Tcf4 is preferentially expressed in murine and human PDCs. Constitutive or inducible deletion of murine E2-2 blocked the development of PDCs but not of other lineages and abolished IFN response to unmethylated DNA. Moreover, E2-2 haploinsufficiency in mice and in human Pitt-Hopkins syndrome patients was associated with aberrant expression profile and impaired IFN response of the PDC. E2-2 directly activated multiple PDC-enriched genes, including transcription factors involved in PDC development (SpiB, Irf8) and function (Irf7). These results identify E2-2 as a specific transcriptional regulator of the PDC lineage in mice and humans and reveal a key function of E proteins in the innate immune system.

Diagnostic yield of various genetic approaches in patients with unexplained developmental delay or mental retardation.

The underlying cause of mental retardation remains unknown in up to 80% of patients. As chromosomal aberrations are the most common known cause of mental retardation, several new methods based on FISH, PCR, and array techniques have been developed over recent years to increase detection rate of subtle aneusomies initially of the gene rich subtelomeric regions, but nowadays also genome wide. As the reported detection rates vary widely between different reports and in order to compare the diagnostic yield of various investigations, we analyzed the diagnostic yield of conventional karyotyping, subtelomeric screening, molecular karyotyping, X‐inactivation studies, and dysmorphological evaluation with targeted laboratory testing in unselected patients referred for developmental delay or mental retardation to our cytogenetic laboratory (n = 600) and to our genetic clinic (n = 570). In the cytogenetic group, 15% of patients showed a disease‐related aberration, while various targeted analyses after dysmorphological investigation led to a diagnosis in about 20% in the genetic clinic group. When adding the patients with a cytogenetic aberration to the patient group seen in genetic clinic, an etiological diagnosis was established in about 40% of the combined study group. A conventional cytogenetic diagnosis was present in 16% of combined patients and a microdeletion syndrome was diagnosed in 5.3%, while subtelomeric screening revealed only 1.3% of causes. Molecular karyotyping with a 10 K SNP array in addition revealed 5% of underlying causes, but 29% of all diagnoses would have been detectable by molecular karyotyping. In those patients without a clear diagnosis, 5.6% of mothers of affected boys showed significant (>95%) skewing of X‐inactivation suggesting X‐linked mental retardation. The most common diagnoses with a frequency of more than 0.5% were Down syndrome (9.2%), common microdeletion 22q11.2 (2.4%), Williams–Beuren syndrome (1.3%), Fragile‐X syndrome (1.2%), Cohen syndrome (0.7%), and monosomy 1p36.3 (0.6%). From our data, we suggest the following diagnostic procedure in patients with unexplained developmental delay or mental retardation: (1) Clinical/dysmorphological investigation with respective targeted analyses; (2) In the remaining patients without an etiological diagnosis, we suggest conventional karyotyping, X‐inactivation screening in mothers of boys, and molecular karyotyping, if available. If molecular karyotyping is not available, subtelomeric screening should be performed.

Mutations in the Pericentrin (PCNT) Gene Cause Primordial Dwarfism

Fundamental processes influencing human growth can be revealed by studying extreme short stature. Using genetic linkage analysis, we find that biallelic loss-of-function mutations in the centrosomal pericentrin (PCNT) gene on chromosome 21q22.3 cause microcephalic osteodysplastic primordial dwarfism type II (MOPD II) in 25 patients. Adults with this rare inherited condition have an average height of 100 centimeters and a brain size comparable to that of a 3-month-old baby, but are of near-normal intelligence. Absence of PCNT results in disorganized mitotic spindles and missegregation of chromosomes. Mutations in related genes are known to cause primary microcephaly (MCPH1, CDK5RAP2, ASPM, and CENPJ).

CNTNAP2 and NRXN1 Are Mutated in Autosomal-Recessive Pitt-Hopkins-like Mental Retardation and Determine the Level of a Common Synaptic Protein in Drosophila

Heterozygous copy-number variants and SNPs of CNTNAP2 and NRXN1, two distantly related members of the neurexin superfamily, have been repeatedly associated with a wide spectrum of neuropsychiatric disorders, such as developmental language disorders, autism spectrum disorders, epilepsy, and schizophrenia. We now identified homozygous and compound-heterozygous deletions and mutations via molecular karyotyping and mutational screening in CNTNAP2 and NRXN1 in four patients with severe mental retardation (MR) and variable features, such as autistic behavior, epilepsy, and breathing anomalies, phenotypically overlapping with Pitt-Hopkins syndrome. With a frequency of at least 1% in our cohort of 179 patients, recessive defects in CNTNAP2 appear to significantly contribute to severe MR. Whereas the established synaptic role of NRXN1 suggests that synaptic defects contribute to the associated neuropsychiatric disorders and to severe MR as reported here, evidence for a synaptic role of the CNTNAP2-encoded protein CASPR2 has so far been lacking. Using Drosophila as a model, we now show that, as known for fly Nrx-I, the CASPR2 ortholog Nrx-IV might also localize to synapses. Overexpression of either protein can reorganize synaptic morphology and induce increased density of active zones, the synaptic domains of neurotransmitter release. Moreover, both Nrx-I and Nrx-IV determine the level of the presynaptic active-zone protein bruchpilot, indicating a possible common molecular mechanism in Nrx-I and Nrx-IV mutant conditions. We therefore propose that an analogous shared synaptic mechanism contributes to the similar clinical phenotypes resulting from defects in human NRXN1 and CNTNAP2.

Haploinsufficiency of TCF4 causes syndromal mental retardation with intermittent hyperventilation (Pitt-Hopkins syndrome).

Pitt-Hopkins syndrome is a rarely reported syndrome of so-far-unknown etiology characterized by mental retardation, wide mouth, and intermittent hyperventilation. By molecular karyotyping with GeneChip Human Mapping 100K SNP arrays, we detected a 1.2-Mb deletion on 18q21.2 in one patient. Sequencing of the TCF4 transcription factor gene, which is contained in the deletion region, in 30 patients with significant phenotypic overlap revealed heterozygous stop, splice, and missense mutations in five further patients with severe mental retardation and remarkable facial resemblance. Thus, we establish the Pitt-Hopkins syndrome as a distinct but probably heterogeneous entity caused by autosomal dominant de novo mutations in TCF4. Because of its phenotypic overlap, Pitt-Hopkins syndrome evolves as an important differential diagnosis to Angelman and Rett syndromes. Both null and missense mutations impaired the interaction of TCF4 with ASCL1 from the PHOX-RET pathway in transactivating an E box-containing reporter construct; therefore, hyperventilation and Hirschsprung disease in patients with Pitt-Hopkins syndrome might be explained by altered development of noradrenergic derivatives.

Human TBX1 missense mutations cause gain of function resulting in the same phenotype as 22q11.2 deletions.

Deletion 22q11.2 syndrome is the most frequent known microdeletion syndrome and is associated with a highly variable phenotype, including DiGeorge and Shprintzen (velocardiofacial) syndromes. Although haploinsufficiency of the T-box transcription factor gene TBX1 is thought to cause the phenotype, to date, only four different point mutations in TBX1 have been reported in association with six of the major features of 22q11.2 deletion syndrome. Although, for the two truncating mutations, loss of function was previously shown, the pathomechanism of the missense mutations remains unknown. We report a novel heterozygous missense mutation, H194Q, in a familial case of Shprintzen syndrome and show that this and the two previously reported missense mutations result in gain of function, possibly through stabilization of the protein dimer DNA complex. We therefore conclude that TBX1 gain-of-function mutations can result in the same phenotypic spectrum as haploinsufficiency caused by loss-of-function mutations or deletions.

Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome.

We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both alleles of POLR1C in three individuals with TCS. These findings identify two additional genes involved in TCS, confirm the genetic heterogeneity of TCS and support the hypothesis that TCS is a ribosomopathy.

Haploinsufficiency of ARID1B, a member of the SWI/SNF-a chromatin-remodeling complex, is a frequent cause of intellectual disability.

Intellectual disability (ID) is a clinically and genetically heterogeneous common condition that remains etiologically unresolved in the majority of cases. Although several hundred diseased genes have been identified in X-linked, autosomal-recessive, or syndromic types of ID, the establishment of an etiological basis remains a difficult task in unspecific, sporadic cases. Just recently, de novo mutations in SYNGAP1, STXBP1, MEF2C, and GRIN2B were reported as relatively common causes of ID in such individuals. On the basis of a patient with severe ID and a 2.5 Mb microdeletion including ARID1B in chromosomal region 6q25, we performed mutational analysis in 887 unselected patients with unexplained ID. In this cohort, we found eight (0.9%) additional de novo nonsense or frameshift mutations predicted to cause haploinsufficiency. Our findings indicate that haploinsufficiency of ARID1B, a member of the SWI/SNF-A chromatin-remodeling complex, is a common cause of ID, and they add to the growing evidence that chromatin-remodeling defects are an important contributor to neurodevelopmental disorders.

Systematic assessment of atypical deletions reveals genotype–phenotype correlation in 22q11.2

Introduction: Clinical variability associated with the common 22q11.2 microdeletion is well known, and has led to a broad application of FISH diagnostics with probes for loci TUPLE1 or D22S75 (N25), although, rarely reported atypical deletions associated with the same phenotypic spectrum would not be discovered by these probes. As most types of 22q11.2 deletions occur between low copy repeats within the region (LCR22), we assumed that atypical deletions should be more common than has been reported. To address this question and the possibility of a deletion size related genotype-phenotype correlation, we systematically assessed the frequency of typical and atypical 22q11.2 deletions in a large cohort of patients. Methods: We used a set of 10 fluorescent in situ hybridisation (FISH) DNA probes, capable of detecting all reported and hypothetical deletions between the LCR22, and analysed 350 patients. Deletion sizes in atypical deletions were established by use of further FISH probes. Frequency of certain atypical deletions was analysed in controls by FISH and quantitative PCR. Results: Patients with conotruncal heart defects (ctCHD) and with typical VCFS phenotype showed the common 3 Mb or nested 1.5 Mb deletions (in 18.5% and 78.6%, respectively), but no atypical deletion, while 5% (3/63) of patients with a mildly suggestive, atypical phenotype showed atypical distal deletions, which were not detected in patients with mental retardation of unknown origin or in healthy controls. Discussion: These statistically significant differences demonstrate that atypical distal 22q11.2 deletions are very uncommon in patients with ctCHDs, while atypical congenital heart defects and mild dysmorphism are recognisable feature of atypical distal deletions. Further phenotype-genotype analysis disclosed association of significant developmental delay with the distal part of the common deletion region, and choanal atresia and atypical CHDs with the adjacent distal deletion region.