Every CFTR variant counts - Target-capture based next-generation-sequencing for molecular diagnosis in the German CF Registry.

BACKGROUND: In times of genotype guided therapy options, a total of 3.2 % of people with CF (pwCF) in the German CF Registry[1] only have one or no CFTR-variant detected after genetic analysis. Additionally, genetic data in the Registry can be documented as free text and can therefore be prone to error. In order to allow the greatest possible amount of pwCF access to modern therapies, we conducted a re-evaluation of free text entries and established a custom-whole-CFTR-locus NGS-approach for all pwCF who remained without genetic confirmation afterwards. METHODS: To this end, we assembled 731 free text variants of 655 pwCF in the German CF Registry. All variants were evaluated using ClinVar, HGMD and CFTR1/2, corrected in the Registries' database and uploaded to ClinVar. PwCF whose diagnosis remained uncertain as well as additional pwCF or pwCFTR-RD that were assembled through a nationwide call for testing of unclear cases were offered genetic analysis. Samples were analysed using a target-capture based NGS-custom-design-panel covering the entire CFTR-locus. RESULTS: Evaluation of free text variants led to the discovery of 43 variants not formerly reported in the context of CF. The Registries' dropdown list was extended by 497 variants and over 500 pwCF were provided with their most up-to-date genotype. Samples of 47 pwCF/pwCFTR-RD were sequenced via NGS with an overall success rate of 61.7 %, resulting in implementation of entire CFTR-genotyping into routine diagnostics. CONCLUSION: Entire CFTR-genotyping can greatly increase the genetic diagnostic rate of pwCF/pwCFTR-RD and should be considered after inconspicuous CFTR screening panels in CFTR-diagnostics.

Differential effects of ELX/TEZ/IVA on organ-specific CFTR function in two patients with the rare CFTR splice mutations c.273+1G>A and c.165-2A>G.

Introduction: Evidence for the efficiency of highly-effective triple-CFTR-modulatory therapy with elexacaftor/tezacaftor/ivacaftor (ETI), either demonstrated in clinical trials or by in vitro testing, is lacking for about 10% of people with cystic fibrosis (pwCF) with rare mutations. Comprehensive assessment of CFTR function can provide critical information on the impact of ETI on CFTR function gains for such rare mutations, lending argument of the prescription of ETI. The mutation c.165-2A>G is a rare acceptor splice mutation that has not yet been functionally characterized. We here describe the functional changes induced by ETI in two brothers who are compound heterozygous for the splice mutations c.273+1G>C and c.165-2A>G. Methods: We assessed the effects of ETI on CFTR function by quantitative pilocarpine iontophoresis (QPIT), nasal potential difference measurements (nPD), intestinal current measurements (ICM), ß-adrenergic sweat secretion tests (SST) and multiple breath washout (MBW) prior to and 4 months after the initiation of ETI. Results: Functional CFTR analysis prior to ETI showed no CFTR function in the respiratory and intestinal epithelia and in the sweat gland reabsorptive duct in either brother. In contrast, ß-adrenergic stimulated, CFTR-mediated sweat secretion was detectable in the CF range. Under ETI, both brothers continued to exhibit high sweat chloride concentration in QPIT, evidence of low residual CFTR function in the respiratory epithelia, but normalized ß-adrenergically stimulated production of primary sweat. Discussion: Our results are the first to demonstrate that the c.165-2A>G/c.273+1G>C mutation genotype permits mutant CFTR protein expression. We showed organ-specific differences in the expression of CFTR and consecutive responses to ETI of the c.165-2A>G/c.273+1G>C CFTR mutants that are probably accomplished by non-canonical CFTR mRNA isoforms. This showcase tells us that the individual response of rare CFTR mutations to highly-effective CFTR modulation cannot be predicted from assays in standard cell cultures, but requires the personalized multi-organ assessment by CFTR biomarkers.

The hair collar sign - a possible indication of cranial dysraphism.

The presence of a circumscribed area of alopecia on the scalp raises several differential diagnostic considerations. A ring of hypertrichosis around such a lesion is called "hair collar sign" and raises suspicion of ectopic neural tissue and an underlying defect of the skull. We report two cases of male newborns with a hair collar surrounding a small localized area of scalp alopecia. In one child a cephalocele was diagnosed radiologically. In the other child an osseous defect was ruled out, and the histological examination confirmed the diagnosis of rudimentary meningocele after complete excision. The importance of this clinical sign has not been addressed in the German dermatological literature.

Undifferentiated murine embryonic stem cells cannot induce portal tolerance but may possess immune privilege secondary to reduced major histocompatibility complex antigen expression.

Induction of donor-specific tolerance using embryonic stem (ES) cells followed by transplantation of ES cell-derived tissues from the same allogeneic strain could theoretically engender successful transplantation without immunosuppression. We sought to induce tolerance using bona fide murine ES cells in immunocompetent mice. ES cells were evaluated for the expression of markers restricted to undifferentiated cells [stage-specific embryonic antigen-1 (SSEA-1) and OCT-4] and the ability to form teratomas in immunodeficient mice. BALB/cByJ mice underwent intraportal inoculation with YC5-EYFP ES cells (129 strain; R1-derived) or saline followed by transplantation with 129X1/SvJ, CBA/J, or BALB/cByJ nonvascularized, neonatal cardiac grafts. Mice were sacrificed at graft failure and underwent histologic evaluation of transplanted grafts and lymphoid organs. ES cells and early differentiated progeny underwent real time (RT)-PCR and fluorescence-activated cell sorting (FACS) analysis to detect major histocompatibility complex (MHC) gene transcription and antigen expression. ES cells expressed markers restricted to undifferentiated cells while maintaining the ability to form teratomas in immunodeficient mice. No prolongation of allograft survival or evidence of lymphoid chimerism was observed in immunocompetent recipient mice despite hepatic teratoma formation. MHC class I, class II, and nonclassical antigens were undetectable on ES cells and early differentiated progeny despite the presence of mRNA transcripts. Class I expression was strongly upregulated upon exposure to gamma-interferon. Intraportal inoculation with murine ES cells does not produce lymphoid chimerism or induce donor-specific unresponsiveness to neonatal cardiac grafts in unmanipulated immunocompetent hosts. However, specific differentiated cell types such as ES cellderived dendritic cells, or alternate routes of ES cell administration, may be effective. ES cells appear to have immune privilege, allowing them to form teratomas in immunocompetent mice.