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Orofacial clefting (OFC) is a frequent congenital anomaly and can occur either in the context of underlying syndromes or in isolation (nonsyndromic). The two common OFC phenotypes are cleft lip with/without cleft palate (CL/P) and cleft palate only (CPO). In this study, we searched for penetrant CL/P genes, by evaluating de novo copy number variants (CNV) from an exome sequencing dataset of 50 nonsyndromic patient-parent trios. We detected a heterozygous 86 kb de novo deletion affecting exons 4-11 of ZFHX4, a gene previously associated with OFC. Genetic and phenotypic data from our in-house and the AGORA cohort (710 and 229 individuals with nonsyndromic CL/P) together with literature and database reviews demonstrate that ZFHX4 variants can lead to both nonsyndromic and syndromic forms not only of CL/P but also CPO. Expression analysis in published single-cell RNA-sequencing data (mouse embryo, zebrafish larva) at relevant time-points support an important role of Zfhx4/zfhx4 in craniofacial development. To characterize the role of zfhx4 in zebrafish craniofacial development, we knocked out/down the zebrafish orthologue. Cartilage staining of the zfhx4 CRISPR F0 knockout and morpholino knockdown at 4 days post-fertilization showed an underdeveloped and abnormally shaped ethmoid plate and cartilaginous jaw (resembling micrognathia). While there is evidence for the dominant inheritance of ZFHX4 variants in OFC, we here present a patient with a possible recessive inheritance. In conclusion, ZFHX4 has a highly heterogeneous phenotypic spectrum and variable mode of inheritance. Our data highlight that ZFHX4 should be considered in genetic testing in patients with nonsyndromic clefting., Competing Interests: Competing interests: For the sake of completeness, we declare that D.D. provides compensated consulting services outside of academia as an independent consultant. Furthermore, K.U.L. is a co-founder of LAMPseq Diagnostics GmbH. Although past and current clients might include biotech, life science, and pharma companies, the services are not related to the presented work and all authors are unaware of any possible competing interests. Ethical approval: Written informed consent was obtained from all participants, or the respective parents/legal guardians, prior to inclusion. The study was approved by the ethics committee of the Medical Faculty of the University of Bonn (ethics approval number 295/14, last dated June 20th, 2022). Animal husbandry and experimental setups were in accordance with European Legislation for the Protection of Animals used for Scientific Purposes (Directive 2010/62/EU). National law exempts all zebrafish experiments conducted at larval stages up to 5 dpf, before larvae begin to feed, from ethical approval., (© 2024. The Author(s).)

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is one of the most common birth defects and has a multifactorial etiology. To date, over 45 loci harboring common risk variants have been identified. However, the effector genes at these loci, and the cell types that are affected by risk alleles, remain largely unknown. To address this, we combined genetic data from an nsCL/P genome-wide association study (GWAS) with single-cell RNA sequencing data obtained from the heads of unaffected human embryos. Using the recently developed single-cell disease relevance score (scDRS) approach, we identified two major cell types involved in nsCL/P development, namely the epithelium and the HAND2+ pharyngeal arches (PA). Combining scDRS with co-expression networks and differential gene expression analysis, we prioritized nsCL/P candidate genes, some of which were additionally supported by GWAS data (e.g., CTNND1, PRTG, RPL35A, RAB11FIP1, KRT19). Our results suggest that specific epithelial and PA sub-cell types are involved in nsCL/P development, and harbor a substantial fraction of the genetic risk for nsCL/P., (© 2024. The Author(s).)

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a frequent congenital malformation with multifactorial etiology. While recent genome-wide association studies (GWAS) have identified several nsCL/P risk loci, the functional effects of the associated non-coding variants are largely unknown. Furthermore, additional risk loci remain undetected due to lack of power. As genetic variants might alter binding of transcription factors (TF), we here hypothesized that the integration of data from TF binding sites, expression analyses and nsCL/P GWAS might help to (i) identify functionally relevant variants at GWAS loci, and (ii) highlight novel risk variants that have been previously undetected. Analysing the craniofacial TF TFAP2A in human embryonic palatal mesenchyme (HEPM) cells, we identified 2845 TFAP2A ChIP-seq peaks, several of which were located near nsCL/P candidate genes (e.g. MSX1 and SPRY2). Comparison with independent data suggest that 802 of them might be specific to craniofacial development, and genes near these peaks are enriched in processes relevant to nsCL/P. Integration with nsCL/P GWAS data, however, did not show robust evidence for co-localization of common nsCL/P risk variants with TFAP2A ChIP-seq peaks. This data set represents a new resource for the analyses of craniofacial processes, and similar approaches with additional cell lines and TFs could be applied to generate further insights into nsCL/P etiology., (© 2022. The Author(s).)

We aimed to identify novel deletions and variants of TP63 associated with orofacial clefting (OFC). Copy number variants were assessed in three OFC families using microarray analysis. Subsequently, we analyzed TP63 in a cohort of 1072 individuals affected with OFC and 706 population-based controls using molecular inversion probes (MIPs). We identified partial deletions of TP63 in individuals from three families affected with OFC. In the OFC cohort, we identified several TP63 variants predicting to cause loss-of-function alleles, including a frameshift variant c.569_576del (p.(Ala190Aspfs*5)) and a nonsense variant c.997C>T (p.(Gln333*)) that introduces a premature stop codon in the DNA-binding domain. In addition, we identified the first missense variants in the oligomerization domain c.1213G>A (p.(Val405Met)), which occurred in individuals with OFC. This variant was shown to abrogate oligomerization of mutant p63 protein into oligomeric complexes, and therefore likely represents a loss-of-function allele rather than a dominant-negative. All of these variants were inherited from an unaffected parent, suggesting reduced penetrance of such loss-of-function alleles. Our data indicate that loss-of-function alleles in TP63 can also give rise to OFC as the main phenotype. We have uncovered the dosage-dependent functions of p63, which were previously rejected.

Macular and cone/cone-rod dystrophies (MD/CCRD) demonstrate a broad genetic and phenotypic heterogeneity, with retinal alterations solely or predominantly involving the central retina. Targeted next-generation sequencing (NGS) is an efficient diagnostic tool for identifying mutations in patient with retinitis pigmentosa, which shows similar genetic heterogeneity. To detect the genetic causes of disease in patients with MD/CCRD, we implemented a two-tier procedure consisting of Sanger sequencing and targeted NGS including genes associated with clinically overlapping conditions. Disease-causing mutations were identified in 74% of 251 consecutive MD/CCRD patients (33% of the variants were novel). Mutations in ABCA4, PRPH2 and BEST1 accounted for 57% of disease cases. Further mutations were identified in CDHR1, GUCY2D, PROM1, CRX, GUCA1A, CERKL, MT-TL1, KIF11, RP1L1, MERTK, RDH5, CDH3, C1QTNF5, CRB1, JAG1, DRAM2, POC1B, NPHP1 and RPGR. We provide detailed illustrations of rare phenotypes, including autofluorescence and optical coherence tomography imaging. Targeted NGS also identified six potential novel genotype-phenotype correlations for FAM161A, INPP5E, MERTK, FBLN5, SEMA4A and IMPDH1. Clinical reassessment of genetically unsolved patients revealed subgroups with similar retinal phenotype, indicating a common molecular disease cause in each subgroup.

It has been shown previously that homozygous and compound-heterozygous variants affecting protein function in the human NLRP genes impact reproduction and/or fetal imprinting patterns. These variants represent so-called 'maternal effect mutations', that is, although female variant carriers are healthy, they are at risk of reproductive failure, and their offspring may develop aberrant methylation and imprinting disorders. In contrast, the relevance to reproductive failure of maternal heterozygous NLRP7 variants remains unclear. The present report describes the identification of a heterozygous NLRP7 variant in a healthy 28-year-old woman with a history of recurrent reproductive failure, and the molecular findings in two of the deceased offspring. Next-generation sequencing (NGS) for NLRP variants was performed. In the tissues of two offspring (one fetus; one deceased premature neonate) methylation of imprinted loci was tested using methylation-specific assays. Both pregnancies had been characterized by the presence of elevated human chorionic gonadotropin (hCG) levels and ovarian cysts. In the mother, a heterozygous nonsense 2-bp deletion in exon 5 of the NLRP7 gene was identified (NM_001127255.1:c.2010_2011del, p.(Phe671Glnfs*18)). In the two investigated offspring, heterogeneous aberrant methylation patterns were detected at imprinted loci. The present data support the hypothesis that heterozygous NLRP7 variants contribute to reproductive wastage, and that these variants represent autosomal dominant maternal effect variants which lead to aberrant imprinting marks in the offspring. Specific screening and close prenatal monitoring of NLRP7 variant carriers is proposed. Egg donation might facilitate successful pregnancy in heterozygous NLRP7 variant carriers.

Parent-of-origin (PofO) effects, such as imprinting are a phenomenon where the effect of variants depends on parental origin. Conventional association studies assume that phenotypic effects are independent of parental origin, and are thus severely underpowered to detect such non-Mendelian effects. Risk of orofacial clefts is influenced by genetic and environmental effects, the latter including maternal-specific factors such as perinatal smoking and folate intake. To identify variants showing PofO effects in orofacial clefts we have used a modification of the family-based transmission disequilibrium test to screen for biased transmission from mothers and fathers to affected offspring, biased ratios of maternal versus paternal transmission, and biased frequencies of reciprocal classes of heterozygotes among offspring. We applied these methods to analyze published genome-wide single-nucleotide polymorphism (SNP) data from ∼2500 trios mainly of European and Asian ethnicity with non-syndromic orofacial clefts, followed by analysis of 64 candidate SNPs in a replication cohort of ∼1200 trios of European origin. In our combined analysis, we did not identify any SNPs achieving conventional genome-wide significance (P<5 × 10(-8)). However, we observed an overall excess of loci showing maternal versus paternal transmission bias (P=0.013), and identified two loci that showed nominally significant effects in the same direction in both the discovery and replication cohorts, raising the potential for PofO effects. These include a possible maternal-specific transmission bias associated with rs12543318 at 8q21.3, a locus identified in a recent meta-analysis of non-syndromic cleft (maternal-specific P=1.5 × 10(-7), paternal-specific P=0.17). Overall, we conclude from this analysis that there are subtle hints of PofO effects in orofacial clefting.

Recent genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with non-syndromic cleft lip with or without cleft palate (NSCL/P), and other previous studies showed distinctly differing facial distance measurements when comparing unaffected relatives of NSCL/P patients with normal controls. Here, we test the hypothesis that genetic loci involved in NSCL/P also influence normal variation in facial morphology. We tested 11 SNPs from 10 genomic regions previously showing replicated evidence of association with NSCL/P for association with normal variation of nose width and bizygomatic distance in two cohorts from Germany (N=529) and the Netherlands (N=2497). The two most significant associations found were between nose width and SNP rs1258763 near the GREM1 gene in the German cohort (P=6 × 10(-4)), and between bizygomatic distance and SNP rs987525 at 8q24.21 near the CCDC26 gene (P=0.017) in the Dutch sample. A genetic prediction model explained 2% of phenotype variation in nose width in the German and 0.5% of bizygomatic distance variation in the Dutch cohort. Although preliminary, our data provide a first link between genetic loci involved in a pathological facial trait such as NSCL/P and variation of normal facial morphology. Moreover, we present a first approach for understanding the genetic basis of human facial appearance, a highly intriguing trait with implications on clinical practice, clinical genetics, forensic intelligence, social interactions and personal identity.

We describe the detailed clinical and molecular characterization of three patients (aged 7, 8(4/12) and 31 years) with overlapping microdeletions in 19p13.12, extending to 19p13.13 in two cases. The patients share the following clinical features with a recently reported 10-year-old girl with a 19p13.12 microdeletion: mental retardation (MR), psychomotor and language delay, hearing impairment, brachycephaly, anteverted nares and ear malformations. All patients share a 359-kb deleted region in 19p13.12 harboring six genes (LPHN1, DDX39, CD97, PKN1, PTGER1 and GIPC1), several of which may be MR candidates because of their function and expression pattern. LPHN1 and PKN1 are the most appealing; LPHN1 for its interaction with Shank family proteins, and PKN1 because it is involved in a variety of functions in neurons, including cytoskeletal organization. Haploinsufficiency of GIPC1 may contribute to hearing impairment for its interaction with myosin VI. A behavioral phenotype was observed in all three patients; it was characterized by overactive disorder associated with MR and stereotyped movements (ICD10) in one patient and hyperactivity in the other two. As Ptger1-null mice show behavioral inhibition and impulsive aggression with defective social interaction, PTGER1 haploinsufficiency may be responsible for the behavioral traits observed in these patients.

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by a deficiency in DNA mismatch repair in consequence of germline mutations mainly in the genes MSH2 and MLH1. Around 10% of patients suspected of HNPCC are identified with large genomic deletions that cannot be detected by conventional methods of mutation screening. The recently developed multiplex ligation-dependent probe amplification (MLPA) proved to be an easy to perform method for deletion detection and is reliable when more than one exon is deleted. We show that, in some cases, apparent deletions of single exons may actually result from single base substitutions or small insertions/deletions in the hybridisation sequence of MLPA probes. We conclude that single exon deletions, detected by MLPA or multiplex PCR, should be validated with additional methods.

A predominance of de novo mutations in the paternal germ line has been reported for several disorders; however, in familial adenomatous polyposis (FAP), the parental origin of APC mutations has been scarcely analysed so far. Among 563 unrelated FAP families with known family history, we identified 58 patients with a suspected de novo mutation in the APC gene. A germline mutation was detected in 52 of them; in 38 patients, the mutation could be excluded in both parents. The five base pair deletion at codon 1309 (c.3927&#95;3931delAAAGA) was over-represented in the group of patients with suspected de novo mutations (17/58=29%), when compared to the group of familial cases (26/505=5%); thus, the high frequency of this mutation is not due to a founder effect but rather due to de novo mutation events. Parental origin of de novo mutations could be traced in 16 families, including three families with large chromosomal deletions. Four mutations were of maternal and 12 of paternal origin, pointing to a moderate preponderance towards paternal origin. Sex-related differences of mutation types could be observed: large deletions and single-base substitutions were exclusively of paternal origin, whereas the small deletions were equally distributed (maternal/paternal ratio 4:4).

Gene transfer of the costimulatory molecules B7-1 and B7-2 induces a potent antitumor immune response in a variety of tumor models. B cell neoplasms including multiple myeloma (MM) often show little or no expression of B7 antigens; they are therefore a potential target for this approach. To increase the expression of human B7 genes in MM cells, both genes and the neomycin phosphotransferase gene were packaged into recombinant adeno-associated virus vectors (rAAV). The resulting recombinant viruses rAAV/B7-1, rAAV/B7-2 and rAAV/Neo were used to transduce the MM cell lines LP-1 and RPMI 8226. This allowed the transduction of up to 80% of LP-1 cells 4 days after infection with purified rAAV particles. The response of human allogeneic T cells to rAAV/B7-1 and rAAV/B7-2 transduced, gamma-irradiated LP-1 cells was assessed by [3H]thymidine incorporation, by RT-PCR-based detection of immunostimulatory cytokine transcripts and by ELISA quantification of cytokines in the supernatant. Stimulation of T cells with rAAV/B7-1 or rAAV/B7-2 transduced LP-1 cells resulted in an up to 10-fold increase of T cell proliferation when compared with LP-1 cells transduced with rAAV/Neo. Similar results were obtained with RPMI 8226 cells. Both rAAV/B7-1 and rAAV/B7-2 transduced LP-1 cells stimulated the T cell secretion of IL-2 and IFN-gamma. Furthermore, [51Cr] release assays showed that rAAV/B7-1 or rAAV/B7-2 transduced LP-1 cells induced a cytolytic T cell (CTL) response, in contrast to LP-1 cells transduced with rAAV/Neo. In all assays, the effects of rAAV/B7-1 and rAAV/B7-2 were similar. Taken together, the results show that rAAV-mediated transfer of B7 genes into MM cell lines is able to enhance the antitumor T cell response and to elicit a cytolytic T cell response.