Extending the allelic spectrum at noncoding risk loci of orofacial clefting.

Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.

Imputation of orofacial clefting data identifies novel risk loci and sheds light on the genetic background of cleft lip ± cleft palate and cleft palate only.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation.

Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate.

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO.

Novel mutations in LRP6 highlight the role of WNT signaling in tooth agenesis.

PURPOSE: We aimed to identify a novel genetic cause of tooth agenesis (TA) and/or orofacial clefting (OFC) by combining whole-exome sequencing (WES) and targeted resequencing in a large cohort of TA and OFC patients. METHODS: WES was performed in two unrelated patients: one with severe TA and OFC and another with severe TA only. After deleterious mutations were identified in a gene encoding low-density lipoprotein receptor-related protein 6 (LRP6), all its exons were resequenced with molecular inversion probes in 67 patients with TA, 1,072 patients with OFC, and 706 controls. RESULTS: We identified a frameshift (c.4594delG, p.Cys1532fs) and a canonical splice-site mutation (c.3398-2A>C, p.?) in LRP6, respectively, in the patient with TA and OFC and in the patient with severe TA only. The targeted resequencing showed significant enrichment of unique LRP6 variants in TA patients but not in nonsyndromic OFC patients. Of the five variants in patients with TA, two affected the canonical splice site and three were missense variants; all variants segregated with the dominant phenotype, and in one case the missense mutation occurred de novo. CONCLUSION: Mutations in LRP6 cause TA in humans.Genet Med 18 11, 1158-1162.

Alopecia and hypotrichosis as characteristic findings in Woodhouse-Sakati syndrome: report of a family with mutation in the C2orf37 gene.

Woodhouse-Sakati syndrome (WSS) is a rare autosomal recessive disorder characterized by alopecia, hypogonadism, diabetes mellitus, intellectual disability, sensorineural deafness, extrapyramidal signs, and low insulinlike growth factor 1 levels. Inter- and intrafamilial phenotypic variability have been reported. Mutations in the C2orf37 gene cause WSS. The present report describes the clinical signs and symptoms of three affected siblings from a consanguineous Bedouin family from Kuwait. Direct sequencing of the C2orf37 gene revealed that the c.436delC (p.Ala147Hisfs*9) mutation was present in a homozygous state in all affected siblings and in a heterozygous state in the parents and a healthy sister. Nine C2orf37 mutations causing WSS have been identified. This family shared the mutation reported earlier in Saudi families and families of Bedouin tribes from Qatar and Israel. No phenotypic or genotypic correlation has been observed. Despite the great phenotypic variability of WSS, hypotrichosis has been observed in all individuals with WSS reported. This condition has not been reported in the dermatologic literature. WSS should be included in the differential diagnosis of syndromic congenital hypotrichosis.

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies.

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.

Prioritization of non-coding elements involved in non-syndromic cleft lip with/without cleft palate through genome-wide analysis of de novo mutations.

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a highly heritable facial disorder. To date, systematic investigations of the contribution of rare variants in non-coding regions to nsCL/P etiology are sparse. Here, we re-analyzed available whole-genome sequence (WGS) data from 211 European case-parent trios with nsCL/P and identified 13,522 de novo mutations (DNMs) in nsCL/P cases, 13,055 of which mapped to non-coding regions. We integrated these data with DNMs from a reference cohort, with results of previous genome-wide association studies (GWASs), and functional and epigenetic datasets of relevance to embryonic facial development. A significant enrichment of nsCL/P DNMs was observed at two GWAS risk loci (4q28.1 (p = 8 × 10-4) and 2p21 (p = 0.02)), suggesting a convergence of both common and rare variants at these loci. We also mapped the DNMs to 810 position weight matrices indicative of transcription factor (TF) binding, and quantified the effect of the allelic changes in silico. This revealed a nominally significant overrepresentation of DNMs (p = 0.037), and a stronger effect on binding strength, for DNMs located in the sequence of the core binding region of the TF Musculin (MSC). Notably, MSC is involved in facial muscle development, together with a set of nsCL/P genes located at GWAS loci. Supported by additional results from single-cell transcriptomic data and molecular binding assays, this suggests that variation in MSC binding sites contributes to nsCL/P etiology. Our study describes a set of approaches that can be applied to increase the added value of WGS data.

Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores.

BACKGROUND: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. METHODS: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein-protein-interactions), were used for final prioritization. CONCLUSION: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.

Integrative approaches generate insights into the architecture of non-syndromic cleft lip with or without cleft palate.

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is a common congenital facial malformation with a multifactorial etiology. Genome-wide association studies (GWASs) have identified multiple genetic risk loci. However, functional interpretation of these loci is hampered by the underrepresentation in public resources of systematic functional maps representative of human embryonic facial development. To generate novel insights into the etiology of nsCL/P, we leveraged published GWAS data on nsCL/P as well as available chromatin modification and expression data on mid-facial development. Our analyses identified five novel risk loci, prioritized candidate target genes within associated regions, and highlighted distinct pathways. Furthermore, the results suggest the presence of distinct regulatory effects of nsCL/P risk variants throughout mid-facial development and shed light on its regulatory architecture. Our integrated data provide a platform to advance hypothesis-driven molecular investigations of nsCL/P and other human facial defects.

LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding.

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.

Non-Syndromic Cleft Lip with or without Cleft Palate: Genome-Wide Association Study in Europeans Identifies a Suggestive Risk Locus at 16p12.1 and Supports SH3PXD2A as a Clefting Susceptibility Gene.

Non-syndromic cleft lip with or without cleft palate (nsCL/P) ranks among the most common human congenital malformations, and has a multifactorial background in which both exogenous and genetic risk factors act in concert. The present report describes a genome-wide association study (GWAS) involving a total of 285 nsCL/P patients and 1212 controls from the Netherlands and Belgium. Twenty of the 40 previously reported nsC/LP susceptibility loci were replicated, which underlined the validity of this sample. SNV-based analysis of the data identified an as yet unreported suggestive locus at chromosome 16p12.1 (p-value of the lead SNV: 4.17 × 10-7). This association was replicated in two of three patient/control replication series (Central European and Yemeni). Gene analysis of the GWAS data prioritized SH3PXD2A at chromosome 10q24.33 as a candidate gene for nsCL/P. To date, support for this gene as a cleft gene has been restricted to data from zebrafish and a knockout mouse model. The present GWAS was the first to implicate SH3PXD2A in non-syndromic cleft formation in humans. In summary, although performed in a relatively small sample, the present GWAS generated novel insights into nsCL/P etiology.

Deletions and loss-of-function variants in TP63 associated with orofacial clefting.

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.

Nonsyndromic cleft palate: An association study at GWAS candidate loci in a multiethnic sample.

BACKGROUND: Nonsyndromic cleft palate only (nsCPO) is a common and multifactorial form of orofacial clefting. In contrast to successes achieved for the other common form of orofacial clefting, that is, nonsyndromic cleft lip with/without cleft palate (nsCL/P), genome wide association studies (GWAS) of nsCPO have identified only one genome wide significant locus. Aim of the present study was to investigate whether common variants contribute to nsCPO and, if so, to identify novel risk loci. METHODS: We genotyped 33 SNPs at 27 candidate loci from 2 previously published nsCPO GWAS in an independent multiethnic sample. It included: (i) a family-based sample of European ancestry (n = 212); and (ii) two case/control samples of Central European (n = 94/339) and Arabian ancestry (n = 38/231), respectively. A separate association analysis was performed for each genotyped dataset, and meta-analyses were performed. RESULTS: After association analysis and meta-analyses, none of the 33 SNPs showed genome-wide significance. Two variants showed nominally significant association in the imputed GWAS dataset and exhibited a further decrease in p-value in a European and an overall meta-analysis including imputed GWAS data, respectively (rs395572: PMetaEU = 3.16 × 10-4 ; rs6809420: PMetaAll = 2.80 × 10-4 ). CONCLUSION: Our findings suggest that there is a limited contribution of common variants to nsCPO. However, the individual effect sizes might be too small for detection of further associations in the present sample sizes. Rare variants may play a more substantial role in nsCPO than in nsCL/P, for which GWAS of smaller sample sizes have identified genome-wide significant loci. Whole-exome/genome sequencing studies of nsCPO are now warranted.