Emma Peskett

Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations

Aims Recent immunohistochemical studies observed the loss of plakoglobin (PG) from the intercalated disc (ID) as a hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC), suggesting a final common pathway for this disease. However, the underlying molecular processes are poorly understood. Methods and results We have identified novel mutations in the desmosomal cadherin desmocollin 2 (DSC2 R203C, L229X, T275M, and G371fsX378). The two missense mutations (DSC2 R203C and T275M) have been functionally characterized, together with a previously reported frameshift variant (DSC2 A897fsX900), to examine their pathogenic potential towards PGs functions at the ID. The three mutant proteins were transiently expressed in various cellular systems and assayed for expression, processing, localization, and binding to other desmosomal components in comparison to wild-type DSC2a protein. The two missense mutations showed defects in proteolytic cleavage, a process which is required for the functional activation of mature cadherins. In both cases, this is thought to cause a reduction of functional DSC2 at the desmosomes in cardiac cells. In contrast, the frameshift variant was incorporated into cardiac desmosomes; however, it showed reduced binding to PG. Conclusion Despite different modes of action, for all three variants, the reduced ability to provide a ligand for PG at the desmosomes was observed. This is in agreement with the reduced intensity of PG at these structures observed in ARVC patients.

Impaired neutrophil migration and phagocytosis in IRAK-4 deficiency

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X-linked CHARGE-like Abruzzo-Erickson syndrome and classic cleft palate with ankyloglossia result from TBX22 splicing mutations

X‐linked cleft palate (CPX) is caused by mutations in the gene encoding the TBX22 transcription factor and is known to exhibit phenotypic variability, usually involving either a complete, partial or submucous cleft palate, with or without ankyloglossia. This study hypothesized a possible involvement of TBX22 in a family with X‐linked, CHARGE‐like Abruzzo–Erickson syndrome, of unknown etiology. The phenotype extends to additional features including sensorineural deafness and coloboma, which are suggested by the Tbx22 developmental expression pattern but not previously associated in CPX patients. A novel TBX22 splice acceptor mutation (c.593−5T>A) was identified that tracked with the phenotype in this family. A novel splice donor variant (c.767+5G>A) and a known canonical splice donor mutation (c.767+1G>A) affecting the same exon were identified in patients with classic CPX phenotypes and were comparatively analyzed using both in silico and in vitro splicing studies. All three variants were predicted to abolish normal mRNA splicing and an in vitro assay indicated that use of alternative splice sites was a likely outcome. Collectively, the data showed the functional effect of several novel intronic splice site variants but most importantly confirms that TBX22 is the gene underlying Abruzzo–Erickson syndrome, expanding the phenotypic spectrum of TBX22 mutations.

Investigation of the Annexin A5 M2 haplotype in 500 white European couples who have experienced recurrent spontaneous abortion

Annexin A5 is a placental anti-coagulant protein that contains four nucleotide substitutions (M2 haplotype) in its promoter. This haplotype is a risk factor for recurrent spontaneous abortion (RSA). The influence of the M2 haplotype in the gestational timing of spontaneous abortions, paternal risk and relationships with known risk factors were investigated. European couples (n = 500) who had experienced three or more consecutive spontaneous abortions, and two fertile control groups, were selected for this study. The allele frequency of M2 was significantly higher among patients who had experienced early RSA than among controls (P = 0.002). No difference was found between controls and patients who had undergone late spontaneous abortions. No difference was found between patients who had experienced RSA who had a live birth or no live births, or between patients who were positive or negative for known risk factors. Male and female partners in each group had similar allele frequencies of M2. The M2 haplotype is a risk factor for early spontaneous abortions, before the 12th week of gestation, and confers about the same relative risk to carriers of both sexes. Having one or more M2 allele(s) in combination with other risk factors further increases the RSA risk.

Mechanical Properties of Calvarial Bones in a Mouse Model for Craniosynostosis

The mammalian cranial vault largely consists of five flat bones that are joined together along their edges by soft fibrous tissues called sutures. Premature closure of the cranial sutures, craniosynostosis, can lead to serious clinical pathology unless there is surgical intervention. Research into the genetic basis of the disease has led to the development of various animal models that display this condition, e.g. mutant type Fgfr2C342Y/+ mice which display early fusion of the coronal suture (joining the parietal and frontal bones). However, whether the biomechanical properties of the mutant and wild type bones are affected has not been investigated before. Therefore, nanoindentation was used to compare the elastic modulus of cranial bone and sutures in wild type (WT) and Fgfr2C342Y/+mutant type (MT) mice during their postnatal development. Further, the variations in properties with indentation position and plane were assessed. No difference was observed in the elastic modulus of parietal bone between the WT and MT mice at postnatal (P) day 10 and 20. However, the modulus of frontal bone in the MT group was lower than the WT group at both P10 (1.39±0.30 vs. 5.32±0.68 GPa; p<0.05) and P20 (5.57±0.33 vs. 7.14±0.79 GPa; p<0.05). A wide range of values was measured along the coronal sutures for both the WT and MT samples, with no significant difference between the two groups. Findings of this study suggest that the inherent mechanical properties of the frontal bone in the mutant mice were different to the wild type mice from the same genetic background. These differences may reflect variations in the degree of biomechanical adaptation during skull growth, which could have implications for the surgical management of craniosynostosis patients.

Impact of rare variants in ARHGAP29 to the etiology of oral clefts: role of loss‐of‐function vs missense variants

Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is a prevalent, complex congenital malformation. Genome‐wide association studies (GWAS) on NSCL/P have consistently identified association for the 1p22 region, in which ARHGAP29 has emerged as the main candidate gene. ARHGAP29 re‐sequencing studies in NSCL/P patients have identified rare variants; however, their clinical impact is still unclear. In this study we identified 10 rare variants in ARHGAP29, including five missense, one in‐frame deletion, and four loss‐of‐function (LoF) variants, in a cohort of 188 familial NSCL/P cases. A significant mutational burden was found for LoF (Sequence Kernel Association Test, p = 0.0005) but not for missense variants in ARHGAP29, suggesting that only LoF variants contribute to the etiology of NSCL/P. Penetrance was estimated as 59%, indicating that heterozygous LoF variants in ARHGAP29 confer a moderate risk to NSCL/P. The GWAS hits in IRF6 (rs642961) and 1p22 (rs560426 and rs4147811) do not seem to contribute to the penetrance of the phenotype, based on co‐segregation analysis. Our data show that rare variants leading to haploinsufficiency of ARHGAP29 represent an important etiological clefting mechanism, and genetic testing for this gene might be taken into consideration in genetic counseling of familial cases.

Analysis of the Fgfr2C342Y mouse model shows condensation defects due to misregulation of Sox9 expression in prechondrocytic mesenchyme.

ABSTRACT Syndromic craniosynostosis caused by mutations in FGFR2 is characterised by developmental pathology in both endochondral and membranous skeletogenesis. Detailed phenotypic characterisation of features in the membranous calvarium, the endochondral cranial base and other structures in the axial and appendicular skeleton has not been performed at embryonic stages. We investigated bone development in the Crouzon mouse model (Fgfr2C342Y) at pre- and post-ossification stages to improve understanding of the underlying pathogenesis. Phenotypic analysis was performed by whole-mount skeletal staining (Alcian Blue/Alizarin Red) and histological staining of sections of CD1 wild-type (WT), Fgfr2C342Y/+ heterozygous (HET) and Fgfr2C342Y/C342Y homozygous (HOM) mouse embryos from embryonic day (E)12.5-E17.5 stages. Gene expression (Sox9, Shh, Fgf10 and Runx2) was studied by in situ hybridisation and protein expression (COL2A1) by immunohistochemistry. Our analysis has identified severely decreased osteogenesis in parts of the craniofacial skeleton together with increased chondrogenesis in parts of the endochondral and cartilaginous skeleton in HOM embryos. The Sox9 expression domain in tracheal and basi-cranial chondrocytic precursors at E13.5 in HOM embryos is increased and expanded, correlating with the phenotypic observations which suggest FGFR2 signalling regulates Sox9 expression. Combined with abnormal staining of type II collagen in pre-chondrocytic mesenchyme, this is indicative of a mesenchymal condensation defect. An expanded spectrum of phenotypic features observed in the Fgfr2C342Y/C342Y mouse embryo paves the way towards better understanding the clinical attributes of human Crouzon–Pfeiffer syndrome. FGFR2 mutation results in impaired skeletogenesis; however, our findings suggest that many phenotypic aberrations stem from a primary failure of pre-chondrogenic/osteogenic mesenchymal condensation and link FGFR2 to SOX9, a principal regulator of skeletogenesis. Summary: Mutation of FGFR2 causes a misregulation of Sox9, leading to disrupted mesenchymal condensation, and thus skeletal and craniofacial birth defects in mice, with implications for human Crouzon syndrome.

Genetic Analyses in Small-for-Gestational-Age Newborns

Context Small for gestational age (SGA) can be the result of fetal growth restriction, which is associated with perinatal morbidity and mortality. Mechanisms that control prenatal growth are poorly understood. Objective The aim of the current study was to gain more insight into prenatal growth failure and determine an effective diagnostic approach in SGA newborns. We hypothesized that one or more copy number variations (CNVs) and disturbed methylation and sequence variants may be present in genes associated with fetal growth. Design A prospective cohort study of subjects with a low birth weight for gestational age. Setting The study was conducted at an academic pediatric research institute. Patients A total of 21 SGA newborns with a mean birth weight below the first centile and a control cohort of 24 appropriate-for-gestational-age newborns were studied. Interventions Array comparative genomic hybridization, genome-wide methylation studies, and exome sequencing were performed. Main Outcome Measures The numbers of CNVs, methylation disturbances, and sequence variants. Results The genetic analyses demonstrated three CNVs, one systematically disturbed methylation pattern, and one sequence variant explaining SGA. Additional methylation disturbances and sequence variants were present in 20 patients. In 19 patients, multiple abnormalities were found. Conclusion Our results confirm the influence of a large number of mechanisms explaining dysregulation of fetal growth. We concluded that CNVs, methylation disturbances, and sequence variants all contribute to prenatal growth failure. These genetic workups can be an effective diagnostic approach in SGA newborns.

SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20

Mutations in SNX14 cause the autosomal recessive cerebellar ataxia 20 (SCAR20). Mutations generally result in loss of protein although several coding region deletions have also been reported. Patient-derived fibroblasts show disrupted autophagy, but the precise function of SNX14 is unknown. The yeast homolog, Mdm1, functions in endoplasmic reticulum (ER)-lysosome/vacuole inter-organelle tethering, but functional conservation in mammals is still required. Here, we show that loss of SNX14 alters but does not block autophagic flux. In addition, we find that SNX14 is an ER-associated protein that functions in neutral lipid homeostasis and inter-organelle crosstalk. SNX14 requires its N-terminal transmembrane helices for ER localization, while the Phox homology (PX) domain is dispensable for subcellular localization. Both SNX14-mutant fibroblasts and SNX14KO HEK293 cells accumulate aberrant cytoplasmic vacuoles, suggesting defects in endolysosomal homeostasis. However, ER-late endosome/lysosome contact sites are maintained in SNX14KO cells, indicating that it is not a prerequisite for ER-endolysosomal tethering. Further investigation of SNX14- deficiency indicates general defects in neutral lipid metabolism. SNX14KO cells display distinct perinuclear accumulation of filipin in LAMP1-positive lysosomal structures indicating cholesterol accumulation. Consistent with this, SNX14KO cells display a slight but detectable decrease in cholesterol ester levels, which is exacerbated with U18666A. Finally, SNX14 associates with ER-derived lipid droplets (LD) following oleate treatment, indicating a role in ER-LD crosstalk. We therefore identify an important role for SNX14 in neutral lipid homeostasis between the ER, lysosomes and LDs that may provide an early intervention target to alleviate the clinical symptoms of SCAR20.

Mice with endogenous TDP‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.