Karl Hackmann

Mutations in the Glycosylphosphatidylinositol Gene PIGL Cause CHIME Syndrome

CHIME syndrome is characterized by colobomas, heart defects, ichthyosiform dermatosis, mental retardation (intellectual disability), and ear anomalies, including conductive hearing loss. Whole-exome sequencing on five previously reported cases identified PIGL, the de-N-acetylase required for glycosylphosphatidylinositol (GPI) anchor formation, as a strong candidate. Furthermore, cell lines derived from these cases had significantly reduced levels of the two GPI anchor markers, CD59 and a GPI-binding toxin, aerolysin (FLAER), confirming the pathogenicity of the mutations.

Expanding the clinical spectrum associated with defects in CNTNAP2 and NRXN1

BackgroundHeterozygous copy-number and missense variants in CNTNAP2 and NRXN1 have repeatedly been associated with a wide spectrum of neuropsychiatric disorders such as developmental language and autism spectrum disorders, epilepsy and schizophrenia. Recently, homozygous or compound heterozygous defects in either gene were reported as causative for severe intellectual disability.Methods99 patients with severe intellectual disability and resemblance to Pitt-Hopkins syndrome and/or suspected recessive inheritance were screened for mutations in CNTNAP2 and NRXN1. Molecular karyotyping was performed in 45 patients. In 8 further patients with variable intellectual disability and heterozygous deletions in either CNTNAP2 or NRXN1, the remaining allele was sequenced.ResultsBy molecular karyotyping and mutational screening of CNTNAP2 and NRXN1 in a group of severely intellectually disabled patients we identified a heterozygous deletion in NRXN1 in one patient and heterozygous splice-site, frameshift and stop mutations in CNTNAP2 in four patients, respectively. Neither in these patients nor in eight further patients with heterozygous deletions within NRXN1 or CNTNAP2 we could identify a defect on the second allele. One deletion in NRXN1 and one deletion in CNTNAP2 occurred de novo, in another family the deletion was also identified in the mother who had learning difficulties, and in all other tested families one parent was shown to be healthy carrier of the respective deletion or mutation.ConclusionsWe report on patients with heterozygous defects in CNTNAP2 or NRXN1 associated with severe intellectual disability, which has only been reported for recessive defects before. These results expand the spectrum of phenotypic severity in patients with heterozygous defects in either gene. The large variability between severely affected patients and mildly affected or asymptomatic carrier parents might suggest the presence of a second hit, not necessarily located in the same gene.

Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity

Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.

Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2−/−γc−/− mice engrafted with either Tre-transduced primary CD4+ T cells, or Tre-transduced CD34+ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV.

Homozygous loss of CHRNA7 on chromosome 15q13.3 causes severe encephalopathy with seizures and hypotonia.

Homozygous Loss of CHRNA7 on Chromosome 15q13.3 Causes Severe Encephalopathy With Seizures and Hypotonia Volker Endris, Karl Hackmann, Teresa M. Neuhann, Ute Grasshoff, Michael Bonin, Ulrich Haug, Gabriele Hahn, Jens C. Schallner, Evelin Schr€ock, Sigrid Tinschert, Gudrun Rappold, and Ute Moog* Department of Molecular Human Genetics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany Institute of Clinical Genetics, Technical University Dresden, Dresden, Germany Institute of Human Genetics, Medical Genetics T€ubingen, T€ubingen, Germany Center for Child Neurology and Social Pediatrics Maulbronn, Maulbronn, Germany Children’s Hospital, Medical Faculty Carl Gustav Carus, University of Technology, Dresden, Germany Institute of Diagnostic Radiology, Department of Pediatric Radiology, Medical Faculty Carl Gustav Carus, University of Technology, Dresden, Germany Department of Human Genetics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany

Severe forms of Baraitser-Winter syndrome are caused by ACTB mutations rather than ACTG1 mutations.

ACTB and ACTG1 mutations have recently been reported to cause Baraitser–Winter syndrome (BRWS) – a rare condition characterized by ptosis, colobomata, neuronal migration disorder, distinct facial anomalies and intellectual disability. One of the patients carrying an ACTB mutation was previously diagnosed with Fryns–Aftimos syndrome (FAS), which is a rare and severe, multiple congenital anomaly (MCA) syndrome whose symptoms partially overlap with that of BRWS. However, several patients with Fryns–Aftimos were considered not to fit into the ACTB and ACTG1 spectrum because of their severe impairment and additional malformations. We report on three patients who had been diagnosed with FAS. All three patients carry a mutation in the ACTB gene. On the basis of the ACTB mutations and analysis of the clinical findings, we reclassify the diagnosis of these patients as severe BRWS. We suggest that mutations in ACTB cause a distinctly more severe phenotype than ACTG1 mutations, despite the structural similarity of beta- and gamma-actins and their overlapping expression pattern. We expand the spectrum of BRWS and confirm that FAS is not a separate entity but an early and severe manifestation of BRWS.

Clonal evolution including partial loss of human leukocyte antigen genes favoring extramedullary acute myeloid leukemia relapse after matched related allogeneic hematopoietic stem cell transplantation.

Background. Relapse of acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT) leaves few therapeutic options, and mechanisms of immune escape of recurring leukemic cells remain poorly understood. Recently, acquired loss of mismatched human leukocyte antigen (HLA) was demonstrated in patients with AML undergoing haploidentical allogeneic HSCT and was suggested not to occur in HLA-matched HSCT. We hypothesized that this mechanism applies to extramedullary AML relapse which occurs frequently after allogeneic HSCT and might also not be restricted to haploidentical HSCT. Methods. DNA from extramedullary AML relapse after HSCT was compared with bone marrow at diagnosis with array comparative genomic hybridization to investigate relapse-specific genomic aberrations in relapsing AML after allogeneic HSCT. Formalin-fixed, paraffin-embedded tissues from the same points of time were assessed for HLA, major histocompatibility complex class I chain-related gene A, and TAP2 immunohistochemistry staining to assess cell surface expression of deleted loci encoded on chromosome 6p. Results. Array comparative genomic hybridization revealed a partial loss of chromosome 6p in extramedullary myeloid sarcoma relapse of AML after sustained complete remission was achieved through matched related allogeneic HSCT. Among others, a deleted region 6p21.32-p21.33, which included several HLA class I genes, was detected. Conclusions. These results suggest that the loss of HLA class I haplotype also occurs in AML relapse after HLA-matched related HSCT. Partial loss of several HLA class I genes and subsequent reduced presentation of minor histocompatibility antigens and reduced ligation of activating natural killer-cell receptors may explain the loss of graft-versus-leukemia response and extramedullary AML relapse in tissue with reduced immunologic surveillance.

De novo microdeletions of chromosome 6q14.1-q14.3 and 6q12.1-q14.1 in two patients with intellectual disability - further delineation of the 6q14 microdeletion syndrome and review of the literature.

Interstitial 6q deletions can cause a variable phenotype depending on the size and location of the deletion. 6q14 deletions have been associated with intellectual disability and a distinct pattern of minor anomalies, including upslanted palpebral fissures with epicanthal folds, a short nose with broad nasal tip, anteverted nares, long philtrum, and thin upper lip. In this study we describe two patients with overlapping 6q14 deletions presenting with developmental delay and characteristic dysmorphism. Molecular karyotyping using array CGH analysis revealed a de novo 8.9 Mb deletion at 6q14.1-q14.3 and a de novo 11.3 Mb deletion at 6q12.1-6q14.1, respectively. We provide a review of the clinical features of twelve other patients with 6q14 deletions detected by array CGH analysis. By assessing all reported data we could not identify a single common region of deletion. Possible candidate genes in 6q14 for intellectual disability might be FILIP1, MYO6, HTR1B, and SNX14.

Mutations in EXOSC2 are associated with a novel syndrome characterised by retinitis pigmentosa, progressive hearing loss, premature ageing, short stature, mild intellectual disability and distinctive gestalt

Background Retinitis pigmentosa in combination with hearing loss can be a feature of different Mendelian disorders. We describe a novel syndrome caused by biallelic mutations in the ‘exosome component 2’ (EXOSC2) gene. Methods Clinical ascertainment of three similar affected patients followed by whole exome sequencing. Results Three individuals from two unrelated German families presented with a novel Mendelian disorder encompassing childhood myopia, early onset retinitis pigmentosa, progressive sensorineural hearing loss, hypothyroidism, short stature, brachydactyly, recognisable facial gestalt, premature ageing and mild intellectual disability. Whole exome sequencing revealed homozygous or compound heterozygous missense variants in the EXOSC2 gene in all three patients. EXOSC2 encodes the ‘ribosomal RNA-processing protein 4’ (RRP4)—one of the core components of the RNA exosome. The RNA exosome is a multiprotein complex that plays key roles in RNA processing and degradation. Intriguingly, the EXOSC2-associated phenotype shows only minimal overlap with the previously reported diseases associated with mutations in the RNA exosome core component genes EXOSC3 and EXOSC8. Conclusion We report a novel condition that is probably caused by altered RNA exosome function and expands the spectrum of clinical consequences of impaired RNA metabolism.

1.6 Mb deletion in chromosome band 3q29 associated with eye abnormalities

We report on a patient with a de novo microdeletion 3q29 detected by molecular karyotyping using array CGH analysis. The girl displayed microphthalmia and cataract, hyperplastic pyloric stenosis, mild dysmorphic facial features, and developmental delay. Array CGH analysis uncovered a 1.6Mb deletion within chromosome band 3q29, which overlaps with the commonly deleted region in 3q29 microdeletion syndrome. According to published data, none of the patients with deletion 3q29 showed either congenital cataract and microphthalmia, or other ocular features. Our report expands the phenotypic spectrum of the 3q29 microdeletion syndrome by adding structural eye malformations.