Mark T. Handley

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18-interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor (GEF) complex. 12 of these 28 interactions are supported by prior reports and we have directly validated novel interactions with SEC22A, TMCO4 and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites (MCSs), interactors included groups of microtubule/membrane-remodelling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP (emopamil binding protein) is a Δ8-Δ7 sterol isomerase and OSBPL2/ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We find that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Further, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated. Our data demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated “mutation negative” probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA–and by implication rI production—correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA. Nucleotide triphosphate bases containing inosine, ITP and dITP, are continually produced within the cell as a consequence of various essential biosynthetic reactions. The enzyme inosine triphosphate pyrophosphatase (ITPase) scavenges ITP and dITP to prevent their incorporation into RNA and DNA. Here we describe two unrelated families with complete loss of ITPase function as a consequence of disruptive mutations affecting both alleles of ITPA, the gene that encodes this protein. Both of the families have a very distinctive and severe combination of clinical problems, most notably a failure of heart muscle that was lethal in infancy or early childhood. They also have features that are reminiscent of another rare genetic disorder affecting the brain and the eyes called Martsolf syndrome. We could not detect any evidence of dITP accumulation in double-stranded DNA from the nucleus in cells from the affected individuals. A low but detectable level of inosine was present in the circular double-stranded DNA present in mitochondria but this did not have any obvious detrimental effect. The inosine accumulation in RNA was detectable in the patient cells. We made both cellular and animal models that were completely deficient in ITPase. Using these reagents we could show that the highest level of inosine accumulation into RNA was seen in the embryonic mouse heart. In this tissue more than 1 in 400 bases in all RNA in the cell was inosine. In normal tissues inosine is almost undetectable using very sensitive assays. The inosine accumulation did not seem to be having a global effect on the balance of RNA molecules or proteins.

Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Through genetic analysis of 85 unrelated “mutation negative” probands referred with Martsolf syndrome we identified two individuals with different homozygous null mutations in ITPA , the gene encoding inosine triphosphate pyrophosphatase (ITPase). The probands reported here each presented with a lethal and highly distinctive disorder; Martsolf syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rl/dl) into RNA and DNA. In Itpa -null cells, dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA but this was not associated with detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in lymphoblastoid RNA from an affected individual. In Itpa -null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA – and by implication rI production - correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA. Author Summary: Nucleotide triphosphate bases containing inosine, ITP and dITP, are continually produced within the cell as a consequence of various essential biosynthetic reactions. The enzyme inosine triphosphate pyrophosphatase (ITPase) scavenges ITP and dITP to prevent their incorporation into RNA and DNA. Here we describe two unrelated families with complete loss of ITPase function as a consequence of disruptive mutations affecting both alleles of ITPA , the gene that encodes this protein. Both of the families have a very distinctive and severe combination of clinical problems, most notably a failure of heart muscle that was lethal in infancy or early childhood. They also have features of another rare genetic disorder affecting the brain and the eyes called Martsolf syndrome. We could not detect any evidence of dITP accumulation in genomic DNA from the affected individuals. A low but detectable level of inosine was present in the mitochondrial DNA but this did not have any obvious detrimental effect. The inosine accumulation in RNA was detectable in the patient cells. We made both cellular and animal models that were completely deficient in ITPase. Using these reagents we could show that the highest level of inosine accumulation into RNA was seen in the embryonic mouse heart. In this tissue more than 1 in 400 bases in all RNA in the cell was inosine. In normal tissues inosine is almost undetectable using very sensitive assays. The inosine accumulation did not seem to be having a global effect on the balance of RNA molecules or proteins.

Purpose: Peroxisomes perform complex metabolic and catabolic functions essential for normal growth and development. Mutations in 14 genes cause a spectrum of peroxisomal disease in humans. Most recently, PEX11B was associated with an atypical peroxisome biogenesis disorder (PBD) in a single individual. In this study, we identify further PEX11B cases and delineate associated phenotypes. Methods: Probands from three families underwent next generation sequencing (NGS) for diagnosis of a multisystem developmental disorder. Autozygosity mapping was conducted in one affected sibling pair. ExomeDepth was used to identify copy number variants from NGS data and confirmed by dosage analysis. Biochemical profiling was used to investigate the metabolic signature of the condition. Results: All patients presented with bilateral cataract at birth but the systemic phenotype was variable, including short stature, skeletal abnormalities, and dysmorphism—features not described in the original case. Next generation sequencing identified biallelic loss-of-function mutations in PEX11B as the underlying cause of disease in each case (PEX11B c.235C>T p.(Arg79Ter) homozygous; PEX11B c.136C>T p.(Arg46Ter) homozygous; PEX11B c.595C>T p.(Arg199Ter) heterozygous, PEX11B ex1-3 del heterozygous). Biochemical studies identified very low plasmalogens in one patient, whilst a mildly deranged very long chain fatty acid profile was found in another. Conclusions: Our findings expand the phenotypic spectrum of the condition and underscore congenital cataract as the consistent primary presenting feature. We also find that biochemical measurements of peroxisome function may be disturbed in some cases. Furthermore, diagnosis by NGS is proficient and may circumvent the requirement for an invasive skin biopsy for disease identification from fibroblast cells.

Chapters provides overview of pathways of development and reviews of dysmorphic syndromes for which the causative gene has been identified. For each disorder, an analysis of the role of the gene in the relevant developmental pathway is provided, along with the mechanism by which mutations in the gene cause the developmental pathology. Emphasis is placed the developmental roles of genes in the causation of hereditary conditions affecting appearance and function.