Gabrielle R. Wilson

Deletion of the Parkin co-regulated gene causes defects in ependymal ciliary motility and hydrocephalus in the quakingviable mutant mouse

The quakingviable mouse (qkv) is a spontaneous recessive mouse mutant with a deletion of approximately 1.1 Mb in the proximal region of chromosome 17. The deletion affects the expression of three genes; quaking (Qk), Parkin-coregulated gene (Pacrg) and parkin (Park2). The resulting phenotype, which includes dysmyelination of the central nervous system and male sterility, is due to reduced expression of Qk and a complete lack of Pacrg expression, respectively. Pacrg is required for correct development of the spermatozoan flagella, a specialized type of motile cilia. In vertebrates, motile cilia are required for multiple functions related to cellular movement or movement of media over a stationary cell surface. To investigate the potential role of PACRG in motile cilia we analysed qkv mutant mice for evidence of cilial dysfunction. Histological and magnetic resonance imaging analyses demonstrated that qkv mutant mice were affected by acquired, communicating hydrocephalus (HC). Structural analysis of ependymal cilia demonstrated that the 9 + 2 arrangement of axonemal microtubules was intact and that both the density of ciliated cells and cilia length was similar to wild-type littermates. Cilia function studies showed a reduction in ependymal cilial beat frequency and cilial mediated flow in qkv mutant mice compared with wild-type littermate controls. Moreover, transgenic expression of Pacrg was necessary and sufficient to correct this deficit and rescue the HC phenotype in the qkv mutant. This study provides novel in vivo evidence that Pacrg is required for motile cilia function and may be involved in the pathogenesis of human ciliopathies, such as HC, asthenospermia and primary ciliary dyskinesia.

Mutations in SH3PXD2B cause Borrone dermato-cardio-skeletal syndrome

Borrone Dermato-Cardio-Skeletal (BDCS) syndrome is a severe progressive autosomal recessive disorder characterized by coarse facies, thick skin, acne conglobata, dysmorphic facies, vertebral abnormalities and mitral valve prolapse. We identified a consanguineous kindred with a child clinically diagnosed with BDCS. Linkage analysis of this family (BDCS1) identified five regions homozygous by descent with a maximum LOD score of 1.75. Linkage analysis of the family that originally defined BDCS (BDCS3) identified an overlapping linkage peak at chromosome 5q35.1. Sequence analysis identified two different homozygous mutations in BDCS1 and BDCS3, affecting the gene encoding the protein SH3 and PX domains 2B (SH3PXD2B), which localizes to 5q35.1. Western blot analysis of patient fibroblasts derived from affected individuals in both families demonstrated complete loss of SH3PXD2B. Homozygosity mapping and sequence analysis in a second published BDCS family (BDCS2) excluded SH3PXD2B. SH3PXD2B is required for the formation of functional podosomes, and loss-of-function mutations in SH3PXD2B have recently been shown to underlie 7 of 13 families with Frank-Ter Haar syndrome (FTHS). FTHS and BDCS share some overlapping clinical features; therefore, our results demonstrate that a proportion of BDCS and FTHS cases are allelic. Mutations in other gene(s) functioning in podosome formation and regulation are likely to underlie the SH3PXD2B-mutation-negative BDSC/FTHS patients.

Implementation of ironXS: a study of the acceptability and feasibility of genetic screening for hereditary hemochromatosis in high schools.

Delatycki MB, Wolthuizen M, Collins V, Varley E, Craven J, Allen KJ, Aitken MA, Bond L, Lockhart PJ, Wilson GR, Macciocca I, Metcalfe SA. Implementation of ironXS: a study of the acceptability and feasibility of genetic screening for hereditary hemochromatosis in high schools.

ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy

AIMS We identified a novel homozygous truncating mutation in the gene encoding alpha kinase 3 (ALPK3) in a family presenting with paediatric cardiomyopathy. A recent study identified biallelic truncating mutations of ALPK3 in three unrelated families; therefore, there is strong genetic evidence that ALPK3 mutation causes cardiomyopathy. This study aimed to clarify the mutation mechanism and investigate the molecular and cellular pathogenesis underlying ALPK3-mediated cardiomyopathy. METHODS AND RESULTS We performed detailed clinical and genetic analyses of a consanguineous family, identifying a new ALPK3 mutation (c.3792G>A, p.W1264X) which undergoes nonsense-mediated decay in ex vivo and in vivo tissues. Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. Multi-electrode array analysis and calcium imaging demonstrated an extended field potential duration and abnormal calcium handling in mutant contractile cultures. CONCLUSIONS This study validates the genetic evidence, suggesting that mutations in ALPK3 can cause familial cardiomyopathy and demonstrates loss of function as the underlying genetic mechanism. We show that ALPK3-deficient cardiomyocytes derived from pluripotent stem cell models recapitulate the ultrastructural and electrophysiological defects observed in vivo. Analysis of differentiated contractile cultures identified abnormal calcium handling as a potential feature of cardiomyocytes lacking ALPK3, providing functional insights into the molecular mechanisms underlying ALPK3-mediated cardiomyopathy.

Molecular analysis of the PArkin co-regulated gene and association with male infertility

OBJECTIVE To investigate the potential role of PArkin co-regulated gene (PACRG) in human male infertility. DESIGN Case-control study. SETTING Academic reproductive biology department. PATIENT(S) Blood samples were obtained from 610 patients and 156 normal control subjects. INTERVENTION(S) Genomic DNA was used as template for polymerase chain reaction amplification of the PACRG promoter and coding exons. The amplified fragments were tested for DNA sequence variations by direct sequencing and restriction enzyme analysis. MAIN OUTCOME MEASURE(S) Gene structure and sequence alterations of PACRG in infertile male patients. RESULT(S) The structure of PACRG was determined to comprise 5 coding exons, generating a single transcript in the testis which encoded a predicted protein of 257 amino acids. No pathogenic mutations were identified; however, a variant in the promoter of PACRG was shown to be significantly associated with azoospermia, but not oligospermia, in the case-control cohort. CONCLUSION(S) Mutation of PACRG was not identified as a cause of male infertility, but variation in the promoter was demonstrated to be a risk factor associated with azoospermia.

Expanding the phenotypic spectrum of ARID1B-mediated disorders and identification of altered cell-cycle dynamics due to ARID1B haploinsufficiency.

BackgroundMutations in genes encoding components of the Brahma-associated factor (BAF) chromatin remodeling complex have recently been shown to contribute to multiple syndromes characterised by developmental delay and intellectual disability. ARID1B mutations have been identified as the predominant cause of Coffin-Siris syndrome and have also been shown to be a frequent cause of nonsyndromic intellectual disability. Here, we investigate the molecular basis of a patient with an overlapping but distinctive phenotype of intellectual disability, plantar fat pads and facial dysmorphism.Methods/resultsHigh density microarray analysis of the patient demonstrated a heterozygous deletion at 6q25.3, which resulted in the loss of four genes including AT Rich Interactive Domain 1B (ARID1B). Subsequent quantitative real-time PCR analysis revealed ARID1B haploinsufficiency in the patient. Analysis of both patient-derived and ARID1B knockdown fibroblasts after serum starvation demonstrated delayed cell cycle re-entry associated with reduced cell number in the S1 phase. Based on the patient’s distinctive phenotype, we ascertained four additional patients and identified heterozygous de novo ARID1B frameshift or nonsense mutations in all of them.ConclusionsThis study broadens the spectrum of ARID1B associated phenotypes by describing a distinctive phenotype including plantar fat pads but lacking the hypertrichosis or fifth nail hypoplasia associated with Coffin-Siris syndrome. We present the first direct evidence in patient-derived cells that alterations in cell cycle contribute to the underlying pathogenesis of syndromes associated with ARID1B haploinsufficiency.

Expression and localization of the Parkin Co-Regulated Gene in mouse CNS suggests a role in ependymal cilia function

Parkin Co-Regulated Gene (PACRG) is a gene that shares a bi-directional promoter with the Parkinsons disease associated gene parkin. The functional role of PACRG is not well understood, although the gene has been associated with parkinsonian syndromes and more recently with eukaryotic cilia and flagella. We investigated the expression of Pacrg in the mouse brain by in situ hybridization and observed robust expression of Pacrg in the cells associated with the lateral, third and fourth ventricle, in addition to the aqueduct of Sylvius and choroid plexus. For all regions of Pacrg expression identified, strong expression was observed in the newborn period and this was maintained into adulthood. Immunohistochemical analysis showed that Pacrg was a component of the ependymal cells and cilia lining the ventricles. Based on our results and the previous association of PACRG homologues with cilia and flagella, we propose that Pacrg is a component of the ependymal cilia and may play an important role in motile cilia development and/or function in the CNS.

Regional and cellular localisation of Parkin Co-Regulated Gene in developing and adult mouse brain

Parkin Co-Regulated Gene (PACRG) is a novel gene that is oriented in a head-to-head array with parkin, and expression of the two genes is regulated by a shared bi-directional promoter. Mutations in parkin are the most common cause of early-onset autosomal recessive Parkinsons disease, however the function of PACRG and potential role in the pathogenesis of Parkinsons disease are unclear. We generated polyclonal anti-PACRG antisera and performed immunohistochemical analysis of the regional and temporal distribution of Pacrg in the mouse brain. The protein was heterogeneously expressed in neurons throughout the mouse brain, with highest levels observed in the rhombencephalon and mesencephalon. Expression was detectable at 1 week of age, increased to maximal levels by 4 weeks and subsequently declined after 3 months. Comparison of parkin and Pacrg immunohistochemistry demonstrated a correlation of both staining distribution and intensity for the two proteins. These results suggest that the transcriptional co-regulation of Pacrg and parkin leads to a similar regional protein distribution in mouse brain, which may have functional significance for the two proteins.

The emerging role of Rab GTPases in the pathogenesis of Parkinson's disease

The identification of pathogenic mutations in Ras analog in brain 39B (RAB39B) and Ras analog in brain 32 (RAB32) that cause Parkinsons disease (PD) has highlighted the emerging role of protein trafficking in disease pathogenesis. Ras analog in brain (Rab) Guanosine triphosphatase (GTPase) function as master regulators of membrane trafficking, including vesicle formation, movement along cytoskeletal networks, and membrane fusion. Recent studies have linked Rab GTPases with α‐synuclein, Leucine‐rich repeat kinase 2, and Vacuolar protein sorting 35, 3 key proteins in PD pathogenesis. In this review, we discuss the various RAB GTPases associated with PD, current progress in the research, and potential future directions. Investigations into the function of RAB GTPases will likely provide significant insight into the etiology of PD and identify novel therapeutic targets for a currently incurable disease.

ironXS: high-school screening for hereditary haemochromatosis is acceptable and feasible.

As the results of the Human Genome Project are realised, screening for genetic mutations that predispose to preventable disease is becoming increasingly possible. How and where such screening should best be offered are critical, unanswered questions. This study aimed to assess the acceptability and feasibility of genetic screening for preventable disease, using the model of hereditary haemochromatosis, in high-school students. Screening was offered for the HFE C282Y substitution to 17 638 students. Questionnaires were administered at the time of screening (Q1) and approximately 1 month after results were communicated (Q2). Outcomes assessed were uptake of screening, change in scores of validated anxiety, affect and health perception scales from Q1 to Q2, knowledge and iron indices in C282Y homozygous individuals. A total of 5757 (32.6%) students had screening and 28 C282Y-homozygous individuals (1 in 206) were identified, and none of the 27 individuals who had iron indices measures had significant iron overload. There was no significant change in measures of anxiety, affect or health perception in C282Y homozygous or non-homozygous individuals. Over 86% of students answered each of five knowledge questions correctly at Q1. Genetic population-based screening for a preventable disease can be offered in schools in a way that results in minimal morbidity for those identified at high risk of disease. The results of this study are not only relevant for haemochromatosis, but for other genetic markers of preventable disease such as those for cardiovascular disease and cancer.