Luisa Mackenroth

New gain-of-function mutation shows CACNA1D as recurrently mutated gene in autism spectrum disorders and epilepsy

Abstract CACNA1D encodes the pore-forming α1-subunit of Cav1.3, an L-type voltage-gated Ca2+-channel. Despite the recent discovery of two de novo missense gain-of-function mutations in Cav1.3 in two individuals with autism spectrum disorder (ASD) and intellectual disability CACNA1D has not been considered a prominent ASD-risk gene in large scale genetic analyses, since such studies primarily focus on likely-disruptive genetic variants. Here we report the discovery and characterization of a third de novo missense mutation in CACNA1D (V401L) in a patient with ASD and epilepsy. For the functional characterization we introduced mutation V401L into two major C-terminal long and short Cav1.3 splice variants, expressed wild-type or mutant channel complexes in tsA-201 cells and performed whole-cell patch-clamp recordings. Mutation V401L, localized within the channel’s activation gate, significantly enhanced current densities, shifted voltage dependence of activation and inactivation to more negative voltages and reduced channel inactivation in both Cav1.3 splice variants. Altogether, these gating changes are expected to result in enhanced Ca2+-influx through the channel, thus representing a strong gain-of-function phenotype. Additionally, we also found that mutant channels retained full sensitivity towards the clinically available Ca2+ -channel blocker isradipine. Our findings strengthen the evidence for CACNA1D as a novel candidate autism risk gene and encourage experimental therapy with available channel-blockers for this mutation. The additional presence of seizures and neurological abnormalities in our patient define a novel phenotype partially overlapping with symptoms in two individuals with PASNA (congenital primary aldosteronism, seizures and neurological abnormalities) caused by similar Cav1.3 gain-of-function mutations.

Identification and Functional Testing of ERCC2 Mutations in a Multi-national Cohort of Patients with Familial Breast- and Ovarian Cancer.

The increasing application of gene panels for familial cancer susceptibility disorders will probably lead to an increased proposal of susceptibility gene candidates. Using ERCC2 DNA repair gene as an example, we show that proof of a possible role in cancer susceptibility requires a detailed dissection and characterization of the underlying mutations for genes with diverse cellular functions (in this case mainly DNA repair and basic cellular transcription). In case of ERCC2, panel sequencing of 1345 index cases from 587 German, 405 Lithuanian and 353 Czech families with breast and ovarian cancer (BC/OC) predisposition revealed 25 mutations (3 frameshift, 2 splice-affecting, 20 missense), all absent or very rare in the ExAC database. While 16 mutations were unique, 9 mutations showed up repeatedly with population-specific appearance. Ten out of eleven mutations that were tested exemplarily in cell-based functional assays exert diminished excision repair efficiency and/or decreased transcriptional activation capability. In order to provide evidence for BC/OC predisposition, we performed familial segregation analyses and screened ethnically matching controls. However, unlike the recently published RECQL example, none of our recurrent ERCC2 mutations showed convincing co-segregation with BC/OC or significant overrepresentation in the BC/OC cohort. Interestingly, we detected that some deleterious founder mutations had an unexpectedly high frequency of > 1% in the corresponding populations, suggesting that either homozygous carriers are not clinically recognized or homozygosity for these mutations is embryonically lethal. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we demonstrate the complexity of correct interpretation for the discovery of “bonafide” breast cancer susceptibility genes.

Label-free differentiation of human pituitary adenomas by FT-IR spectroscopic imaging.

Fourier transform infrared (FT-IR) spectroscopic imaging has been used to characterize different types of pituitary gland tumors and normal pituitary tissue. Freshly resected tumor tissue from surgery was prepared as thin cryosections and examined by FT-IR spectroscopic imaging. Tissue types were discriminated via k-means cluster analysis and a supervised classification algorithm based on linear discriminant analysis. Spectral classification allowed us to discriminate between tumor and non-tumor cells, as well as between tumor cells that produce human growth hormone (hGH+) and tumor cells that do not produce that hormone (hGH−).The spectral classification was compared and contrasted with a histological PAS and orange G stained image. It was further shown that hGH+ pituitary tumor cells show stronger amide bands than tumor cells that do not produce hGH. This study demonstrates that FT-IR spectroscopic imaging can not only potentially serve as a fast and objective approach for discriminating pituitary gland tumors from normal tissue, but that it can also detect hGH-producing tumor cells.

Interstitial 1q23.3q24.1 deletion in a patient with renal malformation, congenital heart disease, and mild intellectual disability

Interstitial deletions including chromosome region 1q23.3q24.1 are rare. Only eight patients with molecularly characterized deletions have been reported to date. Their phenotype included intellectual disability/developmental delay, growth retardation, microcephaly, congenital heart disease, and renal malformations. We report on a female patient with mild developmental delay, congenital heart disease, and bilateral renal hypoplasia in whom an interstitial de novo deletion of approximately 2.7 Mb in 1q23.3q24.1 was detected by array CGH. This is the smallest deletion described in this region so far. Genotype–phenotype comparison with previously published patients allowed us to propose LMX1A and RXRG as potential candidate genes for intellectual disability, PBX1 as a probable candidate gene for renal malformation, and enabled us to narrow down a chromosome region associated with microcephaly.

PBX1 haploinsufficiency leads to syndromic congenital anomalies of the kidney and urinary tract (CAKUT) in humans

Background Congenital anomalies of the kidney and urinary tract (CAKUT) represent a significant healthcare burden since it is the primary cause of chronic kidney in children. CNVs represent a recurrent molecular cause of CAKUT but the culprit gene remains often elusive. Our study aimed to define the gene responsible for CAKUT in patients with an 1q23.3q24.1 microdeletion. Methods We describe eight patients presenting with CAKUT carrying an 1q23.3q24.1 microdeletion as identified by chromosomal microarray analysis (CMA). Clinical features were collected, especially the renal and urinary tract phenotype, and extrarenal features. We characterised PBX1 expression and localisation in fetal and adult kidneys using quantitative RT-PCR and immunohistochemistry. Results We defined a 276-kb minimal common region (MCR) that only overlaps with the PBX1 gene. All eight patients presented with syndromic CAKUT. CAKUT were mostly bilateral renal hypoplasia (75%). The most frequent extrarenal symptoms were developmental delay and ear malformations. We demonstrate that PBX1 is strongly expressed in fetal kidneys and brain and expression levels decreased in adult samples. In control fetal kidneys, PBX1 was localised in nuclei of medullary, interstitial and mesenchymal cells, whereas it was present in endothelial cells in adult kidneys. Conclusions Our results indicate that PBX1 haploinsufficiency leads to syndromic CAKUT as supported by the Pbx1-null mice model. Correct PBX1 dosage appears to be critical for normal nephrogenesis and seems important for brain development in humans. CMA should be recommended in cases of fetal renal anomalies to improve genetic counselling and pregnancy management.

Pierpont syndrome: report of a new patient

Pierpont syndrome (OMIM #602342) is a rare disorder characterized by developmental delay, characteristic facial gestalt, hearing loss, and abnormal fat distribution in the distal limbs. A specific mutation in TBL1XR1 [c.1337A>G; p.(Tyr446Cys)] has been described recently in six unrelated patients with Pierpont syndrome. We report on a male child with developmental delay, distinctive facial dysmorphic features, dystrophy, and abnormal fat distribution in the feet, in whom we identified the identical TBL1XR1 mutation. This patient also had additional clinical features including microphthalmia, pendular nystagmus, cryptorchidism, dermal sinus, and peripheral joint laxity, which had not been reported previously in association with Pierpont syndrome. This patient corroborates the assumption that Pierpont syndrome is exclusively caused by the specific TBL1XR1 missense mutation p.(Tyr446Cys) and the additional features broaden the phenotypic spectrum of this rare disorder.

Spectrum of genetic variants of BRCA1 and BRCA2 in a German single center study

BackgroundDetermination of mutation status of BRCA1 and BRCA2 has become part of the clinical routine. However, the spectrum of genetic variants differs between populations. The aim of this study was to deliver a comprehensive description of all detected variants.MethodsIn families fulfilling one of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC) criteria for genetic testing, one affected was chosen for analysis. DNA of blood lymphocytes was amplified by PCR and prescreened by DHPLC. Aberrant fragments were sequenced. All coding exons and splice sites of BRCA1 and BRCA2 were analyzed. Screening for large rearrangements in both genes was performed by MLPA.ResultsOf 523 index patients, 121 (23.1%) were found to carry a pathogenic or likely pathogenic (class 4/5) mutation. A variant of unknown significance (VUS) was detected in 73/523 patients (13.9%). Two mutations p.Gln1756Profs*74 and p.Cys61Gly comprised 42.3% (n = 33/78) of all detected pathogenic mutations in BRCA1. Most of the other mutations were unique mutations. The most frequently detected mutation in BRCA2 was p.Val1283Lys (13.9%; n = 6/43). Altogether, 101 different neutral genetic variants were counted in BRCA1 (n = 35) and in BRCA2 (n = 66).ConclusionThe two most frequently detected mutations are founder mutations in Poland and Czech Republic. More similarities seem to be shared with our direct neighbor countries compared to other European countries. For comparison of the extended genotype, a shared database is needed.

BRCA1/2 missense mutations and the value of in-silico analyses

INTRODUCTION The clinical implications of genetic variants in BRCA1/2 in healthy and affected individuals are considerable. Variant interpretation, however, is especially challenging for missense variants. The majority of them are classified as variants of unknown clinical significance (VUS). Computational (in-silico) predictive programs are easy to access, but represent only one tool out of a wide range of complemental approaches to classify VUS. With this single-center study, we aimed to evaluate the impact of in-silico analyses in a spectrum of different BRCA1/2 missense variants. METHODS We conducted mutation analysis of BRCA1/2 in 523 index patients with suspected hereditary breast and ovarian cancer (HBOC). Classification of the genetic variants was performed according to the German Consortium (GC)-HBOC database. Additionally, all missense variants were classified by the following three in-silico prediction tools: SIFT, Mutation Taster (MT2) and PolyPhen2 (PPH2). RESULTS Overall 201 different variants, 68 of which constituted missense variants were ranked as pathogenic, neutral, or unknown. The classification of missense variants by in-silico tools resulted in a higher amount of pathogenic mutations (25% vs. 13.2%) compared to the GC-HBOC-classification. Altogether, more than fifty percent (38/68, 55.9%) of missense variants were ranked differently. Sensitivity of in-silico-tools for mutation prediction was 88.9% (PPH2), 100% (SIFT) and 100% (MT2). CONCLUSION We found a relevant discrepancy in variant classification by using in-silico prediction tools, resulting in potential overestimation and/or underestimation of cancer risk. More reliable, notably gene-specific, prediction tools and functional tests are needed to improve clinical counseling.

Skewed X-inactivation in a family with DLG3-associated X-linked intellectual disability

Mutations in DLG3 are a rare cause of non‐syndromic X‐linked intellectual disability (XLID) (MRX90, OMIM *300189). Only ten DLG3 mutations have been reported to date. The majority of female heterozygous mutation carriers was healthy and had random X‐inactivation patterns. We report on an XLID family with a novel DLG3 mutation. The 12‐year‐old male index patient had moderate intellectual disability (ID) and dysmorphic features. The mutation was also present in four female relatives. A maternal aunt had moderate ID and significantly skewed X‐inactivation favorably inactivating the normal DLG3 allele. The probands healthy mother also had skewed X‐inactivation but in the opposite direction (i.e., inactivation of the mutated allele). Two other female relatives had intermediate cognitive phenotypes and random X‐inactivation. This family broadens the mutational and phenotypical spectrum of DLG3‐associated XLID and demonstrates that heterozygous female mutation carriers can be as severely affected as males. Reports of additional families will be needed to elucidate the causes of unfavorable skewing in female XLID patients.

Novel Mutation in the DKC1 Gene: Neonatal Hoyeraal-Hreidarsson Syndrome As a Rare Differential Diagnosis in Pontocerebellar Hypoplasia, Primary Microcephaly, and Progressive Bone Marrow Failure

Primary microcephaly and severe developmental delay are complex but unspecific signs pointing to various genetic or acquired diseases. A concomitant finding of hematological failure may lead to the differential diagnosis of rare genetic diseases such as chromosome breakage disorders or diseases associated with telomere dysfunction. X-linked Hoyeraal-Hreidarsson syndrome (HHS) is a rare heterogenic disorder characterized by severe neurological impairment and progressive bone marrow failure. The latter represents the main cause of mortality, usually in early childhood. We report on the clinical course of an infant with HHS due to a novel mutation in the DKC1 gene and the particular finding of pontocerebellar hypoplasia.