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The RUNT-related transcription factor RUNX2 plays a critical role in osteoblast differentiation, and alterations to gene dosage cause distinct craniofacial anomalies. Uniquely amongst the RUNT-related family, vertebrate RUNX2 encodes a polyglutamine/polyalanine repeat (Gln 23 -Glu-Ala 17 in humans), with the length of the polyalanine component completely conserved in great apes. Surprisingly, a frequent 6-amino acid deletion polymorphism, p.(Ala84_Ala89)del, occurs in humans (termed 11A allele), and a previous association study (Cuellar et al. Bone 137:115395;2020) reported that the 11A variant was significantly more frequent in non-syndromic sagittal craniosynostosis (nsSag; allele frequency [AF] = 0.156; 95% confidence interval [CI] 0.126-0.189) compared to non-syndromic metopic craniosynostosis (nsMet; AF = 0.068; 95% CI 0.045-0.098). However, the gnomAD v.2.1.1 control population used by Cuellar et al. did not display Hardy-Weinberg equilibrium, hampering interpretation. To re-examine this association, we genotyped the RUNX2 11A polymorphism in 225 individuals with sporadic nsSag as parent-child trios and 164 singletons with sporadic nsMet, restricting our analysis to individuals of European ancestry. We compared observed allele frequencies to the non-transmitted alleles in the parent-child trios, and to the genome sequencing data from gnomAD v.4, which display Hardy-Weinberg equilibrium. Observed AFs (and 95% CI) were 0.076 (0.053-0.104) in nsSag and 0.082 (0.055-0.118) in nsMet, compared with 0.062 (0.042-0.089) in non-transmitted parental alleles and 0.065 (0.063-0.067) in gnomAD v.4.0.0 non-Finnish European control genomes. In summary, we observed a non-significant excess, compared to gnomAD data, of 11A alleles in both nsSag (relative risk 1.18, 95% CI 0.83-1.67) and nsMet (relative risk 1.29, 95% CI 0.87-1.92), but we did not replicate the much higher excess of RUNX2 11A alleles in nsSag previously reported (p = 0.0001)., (© 2024 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Purpose: The etiopathogenesis of coronal nonsyndromic craniosynostosis (cNCS), a congenital condition defined by premature fusion of 1 or both coronal sutures, remains largely unknown., Methods: We conducted the largest genome-wide association study of cNCS followed by replication, fine mapping, and functional validation of the most significant region using zebrafish animal model., Results: Genome-wide association study identified 6 independent genome-wide-significant risk alleles, 4 on chromosome 7q21.3 SEM1-DLX5-DLX6 locus, and their combination conferred over 7-fold increased risk of cNCS. The top variants were replicated in an independent cohort and showed pleiotropic effects on brain and facial morphology and bone mineral density. Fine mapping of 7q21.3 identified a craniofacial transcriptional enhancer (eDlx36) within the linkage region of the top variant (rs4727341; odds ratio [95% confidence interval], 0.48[0.39-0.59]; P = 1.2E-12) that was located in SEM1 intron and enriched in 4 rare risk variants. In zebrafish, the activity of the transfected human eDlx36 enhancer was observed in the frontonasal prominence and calvaria during skull development and was reduced when the 4 rare risk variants were introduced into the sequence., Conclusion: Our findings support a polygenic nature of cNCS risk and functional role of craniofacial enhancers in cNCS susceptibility with potential broader implications for bone health., Competing Interests: Conflict of Interest The authors declare no competing interests in relation to the work described.

Purpose: Enrichment of heterozygous missense and truncating SMAD6 variants was previously reported in nonsyndromic sagittal and metopic synostosis, and interaction of SMAD6 variants with a common polymorphism nearBMP2 (rs1884302) was proposed to contribute to inconsistent penetrance. We determined the occurrence of SMAD6 variants in all types of craniosynostosis, evaluated the impact of different missense variants on SMAD6 function, and tested independently whether rs1884302 genotype significantly modifies the phenotype., Methods: We performed resequencing of SMAD6 in 795 unsolved patients with any type of craniosynostosis and genotyped rs1884302 in SMAD6-positive individuals and relatives. We examined the inhibitory activity and stability of SMAD6 missense variants., Results: We found 18 (2.3%) different rare damaging SMAD6 variants, with the highest prevalence in metopic synostosis (5.8%) and an 18.3-fold enrichment of loss-of-function variants comparedwith gnomAD data (P < 10 -7 ). Combined with eight additional variants, ≥20/26 were transmitted from an unaffected parent but rs1884302 genotype did not predict phenotype., Conclusion: Pathogenic SMAD6 variants substantially increase the risk of both nonsyndromic and syndromic presentations of craniosynostosis, especially metopic synostosis. Functional analysis is important to evaluate missense variants. Genotyping of rs1884302 is not clinically useful. Mechanisms to explain the remarkable diversity of phenotypes associated with SMAD6 variants remain obscure.

Our previous genome-wide association study (GWAS) for sagittal nonsyndromic craniosynostosis (sNCS) provided important insights into the genetics of midline CS. In this study, we performed a GWAS for a second midline NCS, metopic NCS (mNCS), using 215 non-Hispanic white case-parent triads. We identified six variants with genome-wide significance (P ≤ 5 × 10 -8 ): rs781716 (P = 4.71 × 10 -9 ; odds ratio [OR] = 2.44) intronic to SPRY3; rs6127972 (P = 4.41 × 10 -8 ; OR = 2.17) intronic to BMP7; rs62590971 (P = 6.22 × 10 -9 ; OR = 0.34), located ~ 155 kb upstream from TGIF2LX; and rs2522623, rs2573826, and rs2754857, all intronic to PCDH11X (P = 1.76 × 10 -8 , OR = 0.45; P = 3.31 × 10 -8 , OR = 0.45; P = 1.09 × 10 -8 , OR = 0.44, respectively). We performed a replication study of these variants using an independent non-Hispanic white sample of 194 unrelated mNCS cases and 333 unaffected controls; only the association for rs6127972 (P = 0.004, OR = 1.45; meta-analysis P = 1.27 × 10 -8 , OR = 1.74) was replicated. Our meta-analysis examining single nucleotide polymorphisms common to both our mNCS and sNCS studies showed the strongest association for rs6127972 (P = 1.16 × 10 -6 ). Our imputation analysis identified a linkage disequilibrium block encompassing rs6127972, which contained an enhancer overlapping a CTCF transcription factor binding site (chr20:55,798,821-55,798,917) that was significantly hypomethylated in mesenchymal stem cells derived from fused metopic compared to open sutures from the same probands. This study provides additional insights into genetic factors in midline CS.

Background: Craniosynostosis, the premature fusion of one or more cranial sutures, occurs in ∼1 in 2250 births, either in isolation or as part of a syndrome. Mutations in at least 57 genes have been associated with craniosynostosis, but only a minority of these are included in routine laboratory genetic testing., Methods: We used exome or whole genome sequencing to seek a genetic cause in a cohort of 40 subjects with craniosynostosis, selected by clinical or molecular geneticists as being high-priority cases, and in whom prior clinically driven genetic testing had been negative., Results: We identified likely associated mutations in 15 patients (37.5%), involving 14 different genes. All genes were mutated in single families, except for IL11RA (two families). We classified the other positive diagnoses as follows: commonly mutated craniosynostosis genes with atypical presentation ( EFNB1 , TWIST1 ); other core craniosynostosis genes ( CDC45 , MSX2, ZIC1 ); genes for which mutations are only rarely associated with craniosynostosis ( FBN1 , HUWE1 , KRAS , STAT3 ); and known disease genes for which a causal relationship with craniosynostosis is currently unknown ( AHDC1 , NTRK2 ). In two further families, likely novel disease genes are currently undergoing functional validation. In 5 of the 15 positive cases, the (previously unanticipated) molecular diagnosis had immediate, actionable consequences for either genetic or medical management (mutations in EFNB1 , FBN1 , KRAS , NTRK2, STAT3 )., Conclusions: This substantial genetic heterogeneity, and the multiple actionable mutations identified, emphasises the benefits of exome/whole genome sequencing to identify causal mutations in craniosynostosis cases for which routine clinical testing has yielded negative results., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Craniosynostosis, the premature fusion of the cranial sutures, is a heterogeneous disorder with a prevalence of ∼1 in 2,200 (refs. 1,2). A specific genetic etiology can be identified in ∼21% of cases, including mutations of TWIST1, which encodes a class II basic helix-loop-helix (bHLH) transcription factor, and causes Saethre-Chotzen syndrome, typically associated with coronal synostosis. Using exome sequencing, we identified 38 heterozygous TCF12 mutations in 347 samples from unrelated individuals with craniosynostosis. The mutations predominantly occurred in individuals with coronal synostosis and accounted for 32% and 10% of subjects with bilateral and unilateral pathology, respectively. TCF12 encodes one of three class I E proteins that heterodimerize with class II bHLH proteins such as TWIST1. We show that TCF12 and TWIST1 act synergistically in a transactivation assay and that mice doubly heterozygous for loss-of-function mutations in Tcf12 and Twist1 have severe coronal synostosis. Hence, the dosage of TCF12-TWIST1 heterodimers is critical for normal coronal suture development.

Background: Microemboli occur commonly during cardiac surgery in adults, and, when present, increase the risk of neuropsychological deficits. Their incidence and significance during correction of congenital heart disease is unknown. The authors hypothesized that microemboli would occur before bypass with right-to-left cardiac shunts and would also occur in large numbers when the aortic crossclamp was released in children during repair of congenital heart defects., Methods: In 25 children studied with carotid artery Doppler, embolic signals were counted and timed in relation to 13 intraoperative events. Patients were classified as either at high risk (obligate right-to-left shunt or uncorrected transposition of the great arteries) or at low risk (net left-to-right shunt or simple obstructive lesions) for paradoxical (venous to arterial) emboli., Results: The median number of emboli detected was 122 (range, 2-2,664). Forty-two percent of all emboli were detected within 3 min of release of the aortic crossclamp. The high-risk group had significantly more emboli (median, 66; range, 0-116) during the time interval before cardiopulmonary bypass than did the low-risk group (median, 8; range, 0-73), with P < 0.01. There was no significant difference between the high- and low-risk groups in the total number of emboli detected. There was no apparent association between number of emboli and gross neurologic deficits., Conclusions: Microemboli can be detected in the carotid arteries of children undergoing repair of congenital heart disease and are especially prevalent immediately after release of the aortic crossclamp. The role of emboli in causing neurologic injury in children undergoing repair of congenital heart disease remains to be determined.

Human respiratory tract chlamydial infections have been studied in Cambridgeshire for many years, but until recently we have been unable to distinguish between infection with Chlamydia psittaci or Chlamydia pneumoniae (TWAR). In this study, we have employed the micro-immunofluorescence (micro-IF) test for this purpose and to look for the relative incidence of C. psittaci and C. pneumoniae infections in Cambridgeshire. Among 50 patients with community-acquired respiratory tract symptoms whose serum samples had Chlamydia complement fixation test titres greater than or equal to 64, 25 had evidence of recent C. psittaci or C. pneumoniae infection. Nineteen (76%) of the 25 patients had evidence of recent C. psittaci infection and of these 16 (84%) had recently had contact with birds. Six patients (24%) had evidence of recent C. pneumoniae infection, and of these, only two (33%) had recently had contact with birds. While C. psittaci was grown from several of the birds associated with human C. psittaci infection, it was not cultured from any of the birds in contact with the two human C. pneumoniae cases.