PRUNE1: a disease-causing gene for secondary microcephaly

In their Letter to the Editor, Karakaya et al. (2017) present an interesting case report describing the clinical course involving secondary microcephaly of a 3-year-old Turkish boy found to be homozygous for a frameshift mutation in PRUNE1 identified through whole exome sequencing. The child presented with congenital hypotonia, contractures and global developmental delay with respiratory insufficiency and seizures developing in the first year of life. The authors note that the affected child’s head circumference plotted on the 75th centile at birth, and that by 38 months of age he had developed microcephaly. Neuroimaging at 14 months revealed cerebral and cerebellar atrophy consistent with other patients described with Prune syndrome (Karaca et al., 2015; Costain et al., 2017; Zollo et al., 2017). Although the child had abnormal neurology from birth, there was a period of early developmental regression. Peripheral spasticity in the lower extremities and optic atrophy were not documented until 38 months. In addition to the PRUNE1 variant, Karakaya et al. also identified a second homozygous variant in the CCDC14 gene in the Turkish child’s whole exome sequencing data that, while listed to have an allele count of 108 in the current Genome Aggregation Database (gnomAD) release, is notably absent in homozygous fashion (Lek et al., 2016). CCDC14 is known to be expressed in human brain, reported to negatively regulate centriole duplication and interact with proteins previously associated with primary microcephaly (Firat-Karalar et al., 2014). Thus, while it seems likely that the homozygous PRUNE1 variant is primarily responsible for the clinical presentation in the Turkish child, it is impossible to determine whether there may be any phenotypical contribution from this additional homozygous sequence variant. Recently, Costain et al. (2017) described a homozygous consensus splice site variant in PRUNE1 (c.521-2A4G; NM_021222.1) in a 2-year-old Oji-Cre male who presented with congenital hypotonia and talipes, whose head circumference was large at birth ( +3 standard deviations), but by 2 years and 2 months plotted on the 50th centile, with a weight and height on the 95th and 75th centiles, respectively. However, it should be noted that the child’s father is macrocephalic ( +4 standard deviations), the published clinical photographs at 2 years 5 months of age illustrate bitemporal narrowing, a sloping forehead and large ears, consistent with a developing microcephaly, and neuroimaging revealed cortical and cerebellar atrophy. He developed respiratory insufficiency shortly after birth, and infantile spasms in the first year of life (Costain et al., 2017). It remains to be determined how the phenotypical outcomes stemming from proposed loss-of-function mutations defined by Karakaya et al. and Costain et al., relate to missense mutations published by Karaca et al. and also Zollo et al., which are likely to involve at least partial gain-of-function outcomes in PRUNE1 activity. However, as more cases are investigated and published, the phenotype associated with autosomal recessive Prune neurodevelopmental disorder, and the functional outcomes of PRUNE1 mutation, are becoming clearer. It is now apparent that while some patients have a small head at birth and others a head circumference in the normal range, the key component of the microcephaly is that it is progressive, and associated with characteristic neuroimaging findings with a thin or hypoplastic corpus callosum and cortical and cerebellar atrophy developing in early childhood. Although all patients with Prune syndrome described to date are neurologically impaired from birth, there also appears to be a neurodegenerative component with progression of the disorder. In our manuscript, we described clinical overlap of Prune syndrome with the neurodegenerative condition associated with homozygous mutations in TBCD (Zollo et al., 2017). TBCD encodes one of the five tubulin-specific chaperones that are required for a/b-tubulin de novo heterodimer formation and the disorder is characterized by developmental regression, seizures, optic atrophy and secondary microcephaly, cortical atrophy with delayed myelination, cerebellar atrophy and thinned corpus callosum (Edvardson et al., 2016; Flex et al., 2016; Miyake et al., 2016; Pode-Shakked et al., 2017). The neurodegenerative phenotype documented in the Turkish child by Karakaya et al. further demonstrates the similarities with the TBCD disorder and Prune syndrome, and confirms optic atrophy to be a feature of Prune syndrome. Interestingly, it is also becoming clear that respiratory insufficiency is a common feature of Prune syndrome, having been documented by Karakaya et al. and in the Oji-Cre child, as well as the youngest affected Omani child described in our manuscript

Investigation of the Molecular Basis of Three New Disorders of Brain Growth and Development Identified Amongst the Amish

Extremes of brain growth have frequently been associated with impaired neurodevelopment and cognition. A significant contribution to our understanding of the processes involved in brain development has been made by the study of single gene disorders which are rare in the general population, but occur with increased frequency in certain endogamous populations. This project stems from findings of a long-running clinical-genetic program, called ‘Windows of Hope’, based amongst the Amish. The project aims were to clinically characterise three new autosomal recessive disorders of brain growth and development, to define the genes and mutations responsible for each, and to investigate the function of the molecules identified. A final further objective of the project in keeping with the wider aims of the Windows of Hope study was to translate the findings of this research into direct clinical benefits to the families and community involved. A combination of clinical phenotyping, autozygosity and linkage mapping, and functional studies, were used to investigate each disorder, which enabled the identification of the novel disease genes in each case. Chapter three describes the identification of a hypomorphic mutation in PCNA responsible for a novel DNA repair disorder. Thus, although it was considered by many that mutations in PCNA would not be compatible with life due to its crucial role in genomic stability and cell division, this study disproves this notion and describes a molecular ‘missing piece’ in DNA repair spectrum disorders. While the relationship between pervasive developmental disorders and megalencephaly is well described, very few single gene disorders associated with this clinical relationship have been identified. Chapter four documents the discovery of two founder mutations in the KPTN gene associated with such a phenotype. The functional data shows that the encoded wild type molecule (kaptin) associates with dynamic actin cytoskeletal structures in cultured neurons and that the causative mutations result in loss of function perturbing this interaction, defining kaptin as a new molecule which is crucial for normal human brain development and function. Chapter five details the investigation of the eponymously named “Hershberger syndrome”, a disorder originally described by McKusick in the Ohio Amish in 1967. Clinical and genetic studies of affected individuals revealed that the syndrome was comprised of two distinct disorders; Aicardi Goutières syndrome due to mutation in SAMHD1, and a new condition characterised by profound neurological impairment, cerebellar involvement and nephrosis. This condition, renamed “nephrocerebellar syndrome” was found to be caused by homozygous mutations in two closely linked genes, WHAMM and WDR73. The genetic and functional data supported the involvement of both molecules in the disease, suggesting that this is a composite phenotype. The identification and functional characterisation of three new genes responsible for abnormalities in brain growth provides an invaluable insight into disease pathogenesis and also identifies molecular pathways important for normal brain development. This is turn enables clinicians to provide a much needed diagnosis for affected individuals and their families as well as the wider community. Medical Research CouncilNewlife Foundation for Disabled Childre

Dominant mutations of the Notch ligand Jagged1 cause peripheral neuropathy

Notch signaling is a highly conserved intercellular pathway with tightly regulated and pleiotropic roles in normal tissue development and homeostasis. Dysregulated Notch signaling has also been implicated in human disease, including multiple forms of cancer, and represents an emerging therapeutic target. Successful development of such therapeutics requires a detailed understanding of potential on-target toxicities. Here, we identify autosomal dominant mutations of the canonical Notch ligand Jagged1 (or JAG1) as a cause of peripheral nerve disease in 2 unrelated families with the hereditary axonal neuropathy Charcot-Marie-Tooth disease type 2 (CMT2). Affected individuals in both families exhibited severe vocal fold paresis, a rare feature of peripheral nerve disease that can be life-threatening. Our studies of mutant protein posttranslational modification and localization indicated that the mutations (p.Ser577Arg, p.Ser650Pro) impair protein glycosylation and reduce JAG1 cell surface expression. Mice harboring heterozygous CMT2-associated mutations exhibited mild peripheral neuropathy, and homozygous expression resulted in embryonic lethality by midgestation. Together, our findings highlight a critical role for JAG1 in maintaining peripheral nerve integrity, particularly in the recurrent laryngeal nerve, and provide a basis for the evaluation of peripheral neuropathy as part of the clinical development of Notch pathway-modulating therapeutics.This article is freely available via Open Access. Click on the publisher URL to access it via the publisher's site.G1002279/MRC_/Medical Research Council/United Kingdom R25 NS065729/NS/NINDS NIH HHS/United States Z01 AG000949/ImNIH/Intramural NIH HHS/United States R01 NS062869/NS/NINDS NIH HHS/United States F31 NS105404/NS/NINDS NIH HHS/United Statespre-print, post-print, publisher's version/PD

MNS1 variant associated with situs inversus and male infertility

Ciliopathy disorders due to abnormalities of motile cilia encompass a range of autosomal recessive conditions typified by chronic otosinopulmonary disease, infertility, situs abnormalities and hydrocephalus. Using a combination of genome-wide SNP mapping and whole exome sequencing (WES), we investigated the genetic cause of a form of situs inversus (SI) and male infertility present in multiple individuals in an extended Amish family, assuming that an autosomal recessive founder variant was responsible. This identified a single shared (2.34 Mb) region of autozygosity on chromosome 15q21.3 as the likely disease locus, in which we identified a single candidate biallelic frameshift variant in MNS1 [NM_018365.2: c.407_410del; p.(Glu136Glyfs*16)]. Genotyping of multiple family members identified randomisation of the laterality defects in other homozygous individuals, with all wild type or MNS1 c.407_410del heterozygous carriers being unaffected, consistent with an autosomal recessive mode of inheritance. This study identifies an MNS1 variant as a cause of laterality defects and male infertility in humans, mirroring findings in Mns1-deficient mice which also display male infertility and randomisation of left-right asymmetry of internal organs, confirming a crucial role for MNS1 in nodal cilia and sperm flagella formation and function.This article is freely available via Open Access. Click on the Publisher URL to access the full-text

Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM).

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins. In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart. To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired

Single-base substitutions in the CHM promoter as a cause of choroideremia

Although over 150 unique mutations affecting the coding sequence of CHM have been identified in patients with the X-linked chorioretinal disease choroideremia (CHM), no regulatory mutations have been reported, and indeed the promoter has not been defined. Here, we describe two independent families affected by CHM bearing a mutation outside the gene's coding region at position c.-98: C>A and C>T, which segregated with the disease. The male proband of family 1 was found to lack CHM mRNA and its gene product Rab escort protein 1, whereas whole-genome sequencing of an affected male in family 2 excluded the involvement of any other known retinal genes. Both mutations abrogated luciferase activity when inserted into a reporter construct, and by further employing the luciferase reporter system to assay sequences 5′ to the gene, we identified the CHM promoter as the region encompassing nucleotides c.-119 to c.-76. These findings suggest that the CHM promoter region should be examined in patients with CHM who lack coding sequence mutations, and reveals, for the first time, features of the gene's regulation

Datasets of whole cell and mitochondrial oxysterols derived from THP-1, SH-SY5Y and human peripheral blood mononuclear cells using targeted metabolomics

The raw datasets of oxysterol quantifications from whole cell and mitochondrial fractions of THP-1 monocytes and macrophages, neuronal-like SH-SH5Y cells and human peripheral blood mononuclear cells are presented. Oxysterols were quantified using a new liquid chromatography-mass spectrometry (LC-MS) and multiple reaction monitoring analysis published in the article “A quantitative LC-MS/MS method for analysis of mitochondrial-specific oxysterol metabolism” in Redox Biology [1]. This method showed improved extraction efficiency and recovery of mono and dihydroxycholesterols from cellular matrix. The datasets derived from the three cell lines are included in the appendix. These datasets provide new information about the oxysterol distribution in THP-1 monocytes and macrophages, SH-SY5Y cells and peripheral blood mononuclear cells. These datasets can be used as a guide for oxysterol distribution in the three cell lines for future studies, and can used for future method optimization, and for comparison of oxysterol recovery with other analytical techniques

Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.This article is freely available via Open Access. Click on the publisher URL to access it via the publisher's site.We acknowledge funding from Wellcome ( 200990 ). S.E. is a Wellcome Senior Investigator. U.F.P. is supported by a predoctoral fellowship from the Basque Government ( PRE_2018_1_0253 ). M.M.O. is supported by a predoctoral fellowship from the University of the Basque Country ( UPV/EHU, PIF 2018 ). I.J.H. is supported by the Carlos III Health Program ( PI17/00380 ), and País Vasco Department of Health ( 2018111043 ; 2018222031 ). A.S. is supported by the UK Medical Research Council with a Senior Non-Clinical Fellowship ( MC_PC_13029 ). T. Harel is supported by the Israel Science Foundation grant 1663/17 . W.H.Y. is supported by the National Institute of General Medical Sciences of the National Institutes of Health through grant 5 P20 GM103636-07 . J.R.L. is supported by the US National Institute of Neurological Disorders and Stroke ( R35NS105078 ), the National Institute of General Medical Sciences ( R01GM106373 ), and the National Human Genome Research Institute and National Heart Lung and Blood Institute (NHGRI/NHBLI) to the Baylor-Hopkins Center for Mendelian Genomics (BHCMG, UM1 HG006542 ). R.W.T. is supported by the Wellcome Centre for Mitochondrial Research ( 203105/Z/16/Z ), the Medical Research Council (MRC) International Centre for Genomic Medicine in Neuromuscular Disease , Mitochondrial Disease Patient Cohort (UK) ( G0800674 ), the UK NIHR Biomedical Research Centre for Aging and Age-related disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust, the MRC/EPSRC Molecular Pathology Node , The Lily Foundation , and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children . The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003). This study makes use of DECIPHER, which is funded by Wellcome. See Nature PMID: 25533962 or https://www.ddduk.org/access.html for full acknowledgment.pre-print, post-print (6 month embargo