Paediatrics (RCH) - Research Publications
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ItemNo Preview AvailablePathogenic Variants in GPC4 Cause Keipert Syndrome(CELL PRESS, 2019-05-02)Glypicans are a family of cell-surface heparan sulfate proteoglycans that regulate growth-factor signaling during development and are thought to play a role in the regulation of morphogenesis. Whole-exome sequencing of the Australian family that defined Keipert syndrome (nasodigitoacoustic syndrome) identified a hemizygous truncating variant in the gene encoding glypican 4 (GPC4). This variant, located in the final exon of GPC4, results in premature termination of the protein 51 amino acid residues prior to the stop codon, and in concomitant loss of functionally important N-linked glycosylation (Asn514) and glycosylphosphatidylinositol (GPI) anchor (Ser529) sites. We subsequently identified seven affected males from five additional kindreds with novel and predicted pathogenic variants in GPC4. Segregation analysis and X-inactivation studies in carrier females provided supportive evidence that the GPC4 variants caused the condition. Furthermore, functional studies of recombinant protein suggested that the truncated proteins p.Gln506∗ and p.Glu496∗ were less stable than the wild type. Clinical features of Keipert syndrome included a prominent forehead, a flat midface, hypertelorism, a broad nose, downturned corners of mouth, and digital abnormalities, whereas cognitive impairment and deafness were variable features. Studies of Gpc4 knockout mice showed evidence of the two primary features of Keipert syndrome: craniofacial abnormalities and digital abnormalities. Phylogenetic analysis demonstrated that GPC4 is most closely related to GPC6, which is associated with a bone dysplasia that has a phenotypic overlap with Keipert syndrome. Overall, we have shown that pathogenic variants in GPC4 cause a loss of function that results in Keipert syndrome, making GPC4 the third human glypican to be linked to a genetic syndrome.
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ItemNo Preview AvailableRecessive variants in ZNF142 cause a complex neurodevelopmental disorder with intellectual disability, speech impairment, seizures, and dystonia(NATURE PUBLISHING GROUP, 2019-11)PURPOSE: The purpose of this study was to expand the genetic architecture of neurodevelopmental disorders, and to characterize the clinical features of a novel cohort of affected individuals with variants in ZNF142, a C2H2 domain-containing transcription factor. METHODS: Four independent research centers used exome sequencing to elucidate the genetic basis of neurodevelopmental phenotypes in four unrelated families. Following bioinformatic filtering, query of control data sets, and secondary variant confirmation, we aggregated findings using an online data sharing platform. We performed in-depth clinical phenotyping in all affected individuals. RESULTS: We identified seven affected females in four pedigrees with likely pathogenic variants in ZNF142 that segregate with recessive disease. Affected cases in three families harbor either nonsense or frameshifting likely pathogenic variants predicted to undergo nonsense mediated decay. One additional trio bears ultrarare missense variants in conserved regions of ZNF142 that are predicted to be damaging to protein function. We performed clinical comparisons across our cohort and noted consistent presence of intellectual disability and speech impairment, with variable manifestation of seizures, tremor, and dystonia. CONCLUSION: Our aggregate data support a role for ZNF142 in nervous system development and add to the emergent list of zinc finger proteins that contribute to neurocognitive disorders.
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ItemReducing the exome search space for Mendelian diseases using genetic linkage analysis of exome genotypes(BIOMED CENTRAL LTD, 2011)Many exome sequencing studies of Mendelian disorders fail to optimally exploit family information. Classical genetic linkage analysis is an effective method for eliminating a large fraction of the candidate causal variants discovered, even in small families that lack a unique linkage peak. We demonstrate that accurate genetic linkage mapping can be performed using SNP genotypes extracted from exome data, removing the need for separate array-based genotyping. We provide software to facilitate such analyses.
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ItemNeuropathology of childhood-onset basal ganglia degeneration caused by mutation of VAC14(WILEY, 2017-12)OBJECTIVE: To characterize the clinical features and neuropathology associated with recessive VAC14 mutations. METHODS: Whole-exome sequencing was used to identify the genetic etiology of a rapidly progressive neurological disease presenting in early childhood in two deceased siblings with distinct neuropathological features on post mortem examination. RESULTS: We identified compound heterozygous variants in VAC14 in two deceased siblings with early childhood onset of severe, progressive dystonia, and neurodegeneration. Their clinical phenotype is consistent with the VAC14-related childhood-onset, striatonigral degeneration recently described in two unrelated children. Post mortem examination demonstrated prominent vacuolation associated with degenerating neurons in the caudate nucleus, putamen, and globus pallidus, similar to previously reported ex vivo vacuoles seen in the late-endosome/lysosome of VAC14-deficient neurons. We identified upregulation of ubiquitinated granules within the cell cytoplasm and lysosomal-associated membrane protein (LAMP2) around the vacuole edge to suggest a process of vacuolation of lysosomal structures associated with active autophagocytic-associated neuronal degeneration. INTERPRETATION: Our findings reveal a distinct clinicopathological phenotype associated with recessive VAC14 mutations.
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ItemFunctional analysis of a hypomorphic allele shows that MMP14 catalytic activity is the prime determinant of the Winchester syndrome phenotype(OXFORD UNIV PRESS, 2018-08-15)Winchester syndrome (WS, MIM #277950) is an extremely rare autosomal recessive skeletal dysplasia characterized by progressive joint destruction and osteolysis. To date, only one missense mutation in MMP14, encoding the membrane-bound matrix metalloprotease 14, has been reported in WS patients. Here, we report a novel hypomorphic MMP14 p.Arg111His (R111H) allele, associated with a mitigated form of WS. Functional analysis demonstrated that this mutation, in contrast to previously reported human and murine MMP14 mutations, does not affect MMP14's transport to the cell membrane. Instead, it partially impairs MMP14's proteolytic activity. This residual activity likely accounts for the mitigated phenotype observed in our patients. Based on our observations as well as previously published data, we hypothesize that MMP14's catalytic activity is the prime determinant of disease severity. Given the limitations of our in vitro assays in addressing the consequences of MMP14 dysfunction, we generated a novel mmp14a/b knockout zebrafish model. The fish accurately reflected key aspects of the WS phenotype including craniofacial malformations, kyphosis, short-stature and reduced bone density owing to defective collagen remodeling. Notably, the zebrafish model will be a valuable tool for developing novel therapeutic approaches to a devastating bone disorder.
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ItemBioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS(CELL PRESS, 2019-07-03)Genomic technologies such as next-generation sequencing (NGS) are revolutionizing molecular diagnostics and clinical medicine. However, these approaches have proven inefficient at identifying pathogenic repeat expansions. Here, we apply a collection of bioinformatics tools that can be utilized to identify either known or novel expanded repeat sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 families with a clinical diagnosis of cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Analysis of whole-genome sequence (WGS) data with five independent algorithms identified a recessively inherited intronic repeat expansion [(AAGGG)exp] in the gene encoding Replication Factor C1 (RFC1). This motif, not reported in the reference sequence, localized to an Alu element and replaced the reference (AAAAG)11 short tandem repeat. Genetic analyses confirmed the pathogenic expansion in 18 of 22 CANVAS-affected families and identified a core ancestral haplotype, estimated to have arisen in Europe more than twenty-five thousand years ago. WGS of the four RFC1-negative CANVAS-affected families identified plausible variants in three, with genomic re-diagnosis of SCA3, spastic ataxia of the Charlevoix-Saguenay type, and SCA45. This study identified the genetic basis of CANVAS and demonstrated that these improved bioinformatics tools increase the diagnostic utility of WGS to determine the genetic basis of a heterogeneous group of clinically overlapping neurogenetic disorders.
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ItemHeterozygous mutations in HISD17B4 cause juvenile peroxisomal D-bifunctional protein deficiency(LIPPINCOTT WILLIAMS & WILKINS, 2016-12)OBJECTIVE: To determine the genetic cause of slowly progressive cerebellar ataxia, sensorineural deafness, and hypergonadotropic hypogonadism in 5 patients from 3 different families. METHODS: The patients comprised 2 sib pairs and 1 sporadic patient. Clinical assessment included history, physical examination, and brain MRI. Linkage analysis was performed separately on the 2 sets of sib pairs using single nucleotide polymorphism microarrays, followed by analysis of the intersection of the regions. Exome sequencing was performed on 1 affected patient with variant filtering and prioritization undertaken using these intersected regions. RESULTS: Using a combination of sequencing technologies, we identified compound heterozygous mutations in HSD17B4 in all 5 affected patients. In all 3 families, peroxisomal D-bifunctional protein (DBP) deficiency was caused by compound heterozygosity for 1 nonsense/deletion mutation and 1 missense mutation. CONCLUSIONS: We describe 5 patients with juvenile DBP deficiency from 3 different families, bringing the total number of reported patients to 14, from 8 families. This report broadens and consolidates the phenotype associated with juvenile DBP deficiency.
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ItemComplete callosal agenesis, pontocerebellar hypoplasia, and axonal neuropathy due to AMPD2 loss(LIPPINCOTT WILLIAMS & WILKINS, 2015-08)OBJECTIVE: To determine the molecular basis of a severe neurologic disorder in a large consanguineous family with complete agenesis of the corpus callosum (ACC), pontocerebellar hypoplasia (PCH), and peripheral axonal neuropathy. METHODS: Assessment included clinical evaluation, neuroimaging, and nerve conduction studies (NCSs). Linkage analysis used genotypes from 7 family members, and the exome of 3 affected siblings was sequenced. Molecular analyses used Sanger sequencing to perform segregation studies and cohort analysis and Western blot of patient-derived cells. RESULTS: Affected family members presented with postnatal microcephaly and profound developmental delay, with early death in 3. Neuroimaging, including a fetal MRI at 30 weeks, showed complete ACC and PCH. Clinical evaluation showed areflexia, and NCSs revealed a severe axonal neuropathy in the 2 individuals available for electrophysiologic study. A novel homozygous stopgain mutation in adenosine monophosphate deaminase 2 (AMPD2) was identified within the linkage region on chromosome 1. Molecular analyses confirmed that the mutation segregated with disease and resulted in the loss of AMPD2. Subsequent screening of a cohort of 42 unrelated individuals with related imaging phenotypes did not reveal additional AMPD2 mutations. CONCLUSIONS: We describe a family with a novel stopgain mutation in AMPD2. We expand the phenotype recently described as PCH type 9 to include progressive postnatal microcephaly, complete ACC, and peripheral axonal neuropathy. Screening of additional individuals with related imaging phenotypes failed to identify mutations in AMPD2, suggesting that AMPD2 mutations are not a common cause of combined callosal and pontocerebellar defects.
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ItemFamilial cortical dysplasia type IIA caused by a germline mutation in DEPDC5(WILEY, 2015-05)Whole-exome sequencing of two brothers with drug-resistant, early-onset, focal epilepsy secondary to extensive type IIA focal cortical dysplasia identified a paternally inherited, nonsense variant of DEPDC5 (c.C1663T, p.Arg555*). This variant has previously been reported to cause familial focal epilepsy with variable foci in patients with normal brain imaging. Immunostaining of resected brain tissue from both brothers demonstrated mammalian target of rapamycin (mTOR) activation. This report shows the histopathological features of cortical dysplasia associated with a DEPDC5 mutation, confirms mTOR dysregulation in the malformed tissue and expands the spectrum of neurological manifestations of DEPDC5 mutations to include severe phenotypes with large areas of cortical malformation.