FATP4 missense and nonsense mutations cause similar features in Ichthyosis Prematurity Syndrome

Background

Vibrio cholerae O1 and V. cholerae O139 infect humans, causing the diarrheal and waterborne disease cholera, which is a worldwide health problem. V. cholerae and the free-living amoebae Acanthamoeba species are present in aquatic environments, including drinking water and it has shown that Acanthamoebae support bacterial growth and survival. Recently it has shown that Acanthamoeba species enhanced growth and survival of V. cholerae O1 and O139. Water samples from different cholera endemic areas in Sudan were collected with the aim to detect both V. cholerae and Acanthamoeba species from same natural water samples by polymerase chain reaction (PCR).

Findings

For the first time both V. cholerae and Acanthamoeba species were detected in same natural water samples collected from different cholera endemic areas in Sudan. 89% of detected V. cholerae was found with Acanthamoeba in same water samples.

Conclusions

The current findings disclose Acanthamoedae as a biological factor enhancing survival of V. cholerae in nature.     

Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.     

Mutations in CERS3 Cause Autosomal Recessive Congenital Ichthyosis in Humans

Autosomal recessive congenital ichthyosis (ARCI) is a rare genetic disorder of the skin characterized by abnormal desquamation over the whole body. In this study we report four patients from three consanguineous Tunisian families with skin, eye, heart, and skeletal anomalies, who harbor a homozygous contiguous gene deletion syndrome on chromosome 15q26.3. Genome-wide SNP-genotyping revealed a homozygous region in all affected individuals, including the same microdeletion that partially affects two coding genes (ADAMTS17, CERS3) and abolishes a sequence for a long non-coding RNA (FLJ42289). Whereas mutations in ADAMTS17 have recently been identified in autosomal recessive Weill-Marchesani-like syndrome in humans and dogs presenting with ophthalmologic, cardiac, and skeletal abnormalities, no disease associations have been described for CERS3 (ceramide synthase 3) and FLJ42289 so far. However, analysis of additional patients with non-syndromic ARCI revealed a splice site mutation in CERS3 indicating that a defect in ceramide synthesis is causative for the present skin phenotype of our patients. Functional analysis of patient skin and in vitro differentiated keratinocytes demonstrated that mutations in CERS3 lead to a disturbed sphingolipid profile with reduced levels of epidermis-specific very long-chain ceramides that interferes with epidermal differentiation. Taken together, these data present a novel pathway involved in ARCI development and, moreover, provide the first evidence that CERS3 plays an essential role in human sphingolipid metabolism for the maintenance of epidermal lipid homeostasis.     

ELOVL4 Mutations That Cause Spinocerebellar Ataxia-34 Differentially Alter Very Long Chain Fatty Acid Biosynthesis

The FA Elongase-4 (ELOVL4) enzyme mediates biosynthesis of both very long chain (VLC)-PUFAs and VLC-saturated FA (VLC-SFAs). VLC-PUFAs play critical roles in retina and sperm function, whereas VLC-SFAs are predominantly associated with brain function and maintenance of the skin permeability barrier. While some ELOVL4 mutations cause Autosomal Dominant Stargardt-like Macular Dystrophy (STGD3), other ELOVL4 point mutations, such as L168F and W246G, affect the brain and/or skin, leading to Spinocerebellar Ataxia-34 (SCA34) and Erythrokeratodermia variabilis. The mechanisms by which these ELOVL4 mutations alter VLC-PUFA and VLC-SFA biosynthesis to cause the different tissue-specific pathologies are not well understood. To understand how these mutations alter VLC-PUFA and VLC-SFA biosynthesis, we expressed WT-ELOVL4, L168F, and W246G ELOVL4 variants in cell culture and supplemented the cultures with VLC-PUFA or VLC-SFA precursors. Total lipids were extracted, converted to FA methyl esters, and quantified by gas chromatography. We showed that L168F and W246G mutants were capable of VLC-PUFA biosynthesis. W246G synthesized and accumulated 32:6n3, while L168F exhibited gain of function in VLC-PUFA biosynthesis as it made 38:5n3, which we did not detect in WT-ELOVL4 or W246G-expressing cells. However, compared with WT-ELOVL4, both L168F and W246G mutants were deficient in VLC-SFA biosynthesis, especially the W246G protein, which showed negligible VLC-SFA biosynthesis. These results suggest VLC-PUFA biosynthetic capabilities of L168F and W246G in the retina, which may explain the lack of retinal phenotype in SCA34. Defects in VLC-SFA biosynthesis by these variants may be a contributing factor to the pathogenic mechanism of SCA34 and Erythrokeratodermia variabilis.     

Mutations in NOTCH1 Cause Adams-Oliver Syndrome

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5′ region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743−1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.     

TCTN3 Mutations Cause Mohr-Majewski Syndrome

Orofaciodigital syndromes (OFDSs) consist of a group of heterogeneous disorders characterized by abnormalities in the oral cavity, face, and digits and associated phenotypic abnormalities that lead to the delineation of 13 OFDS subtypes. Here, by a combined approach of homozygozity mapping and exome ciliary sequencing, we identified truncating TCTN3 mutations as the cause of an extreme form of OFD associated with bone dysplasia, tibial defect, cystic kidneys, and brain anomalies (OFD IV, Mohr-Majewski syndrome). Analysis of 184 individuals with various ciliopathies (OFD, Meckel, Joubert, and short rib polydactyly syndromes) led us to identify four additional truncating TCTN3 mutations in unrelated fetal cases with overlapping Meckel and OFD IV syndromes and one homozygous missense mutation in a family with Joubert syndrome. By exploring roles of TCTN3 in human ciliary related functions, we found that TCTN3 is necessary for transduction of the sonic hedgehog (SHH) signaling pathway, as revealed by abnormal processing of GLI3 in patient cells. These results are consistent with the suggested role of its murine ortholog, which forms a complex at the ciliary transition zone with TCTN1 and TCTN2, both of which are also implicated in the transduction of SHH signaling. Overall, our data show the involvement of the transition zone protein TCTN3 in the regulation of the key SHH signaling pathway and that its disruption causes a severe form of ciliopathy, combining features of Meckel and OFD IV syndromes.     

Mutations in MED12 Cause X-Linked Ohdo Syndrome

Ohdo syndrome comprises a heterogeneous group of disorders characterized by intellectual disability (ID) and typical facial features, including blepharophimosis. Clinically, these blepharophimosis-ID syndromes have been classified in five distinct subgroups, including the Maat-Kievit-Brunner (MKB) type, which, in contrast to the others, is characterized by X-linked inheritance and facial coarsening at older age. We performed exome sequencing in two families, each with two affected males with Ohdo syndrome MKB type. In the two families, MED12 missense mutations (c.3443G>A [p.Arg1148His] or c.3493T>C [p.Ser1165Pro]) segregating with the phenotype were identified. Upon subsequent analysis of an additional cohort of nine simplex male individuals with Ohdo syndrome, one additional de novo missense change (c.5185C>A [p.His1729Asn]) in MED12 was detected. The occurrence of three different hemizygous missense mutations in three unrelated families affected by Ohdo syndrome MKB type shows that mutations in MED12 are the underlying cause of this X-linked form of Ohdo syndrome. Together with the recently described KAT6B mutations resulting in Ohdo syndrome Say/Barber/Biesecker/Young/Simpson type, our findings point to aberrant chromatin modification as being central to the pathogenesis of Ohdo syndrome.     

Mutations in EMP2 Cause Childhood-Onset Nephrotic Syndrome

Nephrotic syndrome (NS) is a genetically heterogeneous group of diseases that are divided into steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS). SRNS inevitably leads to end-stage kidney disease, and no curative treatment is available. To date, mutations in more than 24 genes have been described in Mendelian forms of SRNS; however, no Mendelian form of SSNS has been described. To identify a genetic form of SSNS, we performed homozygosity mapping, whole-exome sequencing, and multiplex PCR followed by next-generation sequencing. We thereby detected biallelic mutations in EMP2 (epithelial membrane protein 2) in four individuals from three unrelated families affected by SRNS or SSNS. We showed that EMP2 exclusively localized to glomeruli in the kidney. Knockdown of emp2 in zebrafish resulted in pericardial effusion, supporting the pathogenic role of mutated EMP2 in human NS. At the cellular level, we showed that knockdown of EMP2 in podocytes and endothelial cells resulted in an increased amount of CAVEOLIN-1 and decreased cell proliferation. Our data therefore identify EMP2 mutations as causing a recessive Mendelian form of SSNS.     

Fatty Acid Transport Through the Blood-Brain Barrier

Across the cerebral capillaries, the anatomical locus of the blood-brain barrier, the unidirectional influxes of the saturated fatty acids, octanoic and myristic acids, and the unsaturated essential fatty acid, linoleic acid, were measured. Employing an in situ rat brain perfusion technique that allows control of perfusate composition and accurate measurement of perfusate-to-brain fatty acid transport, we found that both [¹⁴C]octanoic and [¹⁴C]myristic acids were transported through the blood-brain barrier in vivo, in large part, by a specific, probenecid-sensitive transport system. However, the transport of [¹⁴C]linoleic acid was not probenecid sensitive. With 0.5 μM fatty acid but no plasma proteins in the perfusate, the permeability-surface area constant was higher for myristic acid (4.8 × 10^--²× s^-1) than for octanoic and linoleic acids (1.5 and 1.2 × 10^--²× s^-1, respectively). Approximately 70, 30, and 25% of the [¹⁴C]myristic, [¹⁴C]octanoic, or [¹⁴C]linoleic acids, respectively, were extracted from the perfusate.     

Novel Mutations of the FASN Gene and Their Effect on Fatty Acid Composition in Japanese Black Beef

Eight novel and four known mutations were detected in the coding sequence of the bovine fatty acid synthase (FASN) gene of an F2 population from Japanese Black and Limousin cattle. Two mutations, g.16024A>G and g.16039T>C, detected in exon 34, which determine amino acid substitutions of threonine (T) to alanine (A) and tryptophan (W) to arginine (R), were clearly separated in the parental breeds. The haplotypes (TW and AR) segregated in F2 individuals and had a significant effect on the fatty acid composition of backfat, intermuscular fat, and intramuscular fat. The TW haplotype was associated with increasing C18:0 and C18:1 content and the ratio of monounsaturated to saturated fatty acids, and decreasing C14:0, C14:1, C16:0, and C16:1 content. The two mutations were screened in two commercial Japanese Black half-sibling populations and similarly determined the contribution to the fatty acid composition of intramuscular fat. The frequency of the TW haplotype was markedly higher in Japanese Black (0.67) than in Holstein (0.17), Angus (0.02), and Hereford (0.07). We conclude that these mutations may contribute to the characteristic fatty acid composition of Japanese Black beef.     

Mutations in CKAP2L,the Human Homolog of the Mouse Radmis Gene,Cause Filippi Syndrome

Filippi syndrome is a rare, presumably autosomal-recessive disorder characterized by microcephaly, pre- and postnatal growth failure, syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals have intellectual disability, seizures, undescended testicles in males, and teeth and hair abnormalities. We performed homozygosity mapping and whole-exome sequencing in a Sardinian family with two affected children and identified a homozygous frameshift mutation, c.571dupA (p.Ile191Asnfs^∗6), in CKAP2L, encoding the protein cytoskeleton-associated protein 2-like (CKAP2L). The function of this protein was unknown until it was rediscovered in mice as Radmis (radial fiber and mitotic spindle) and shown to play a pivotal role in cell division of neural progenitors. Sanger sequencing of CKAP2L in a further eight unrelated individuals with clinical features consistent with Filippi syndrome revealed biallelic mutations in four subjects. In contrast to wild-type lymphoblastoid cell lines (LCLs), dividing LCLs established from the individuals homozygous for the c.571dupA mutation did not show CKAP2L at the spindle poles. Furthermore, in cells from the affected individuals, we observed an increase in the number of disorganized spindle microtubules owing to multipolar configurations and defects in chromosome segregation. The observed cellular phenotypes are in keeping with data from in vitro and in vivo knockdown studies performed in human cells and mice, respectively. Our findings show that loss-of-function mutations in CKAP2L are a major cause of Filippi syndrome.     

Mutations in PIK3R1 Cause SHORT Syndrome

SHORT syndrome is a rare, multisystem disease characterized by short stature, anterior-chamber eye anomalies, characteristic facial features, lipodystrophy, hernias, hyperextensibility, and delayed dentition. As part of the FORGE (Finding of Rare Disease Genes) Canada Consortium, we studied individuals with clinical features of SHORT syndrome to identify the genetic etiology of this rare disease. Whole-exome sequencing in a family trio of an affected child and unaffected parents identified a de novo frameshift insertion, c.1906_1907insC (p.Asn636Thrfs^∗18), in exon 14 of PIK3R1. Heterozygous mutations in exon 14 of PIK3R1 were subsequently identified by Sanger sequencing in three additional affected individuals and two affected family members. One of these mutations, c.1945C>T (p.Arg649Trp), was confirmed to be a de novo mutation in one affected individual and was also identified and shown to segregate with the phenotype in an unrelated family. The other mutation, a de novo truncating mutation (c.1971T>G [p.Tyr657^∗]), was identified in another affected individual. PIK3R1 is involved in the phosphatidylinositol 3 kinase (PI3K) signaling cascade and, as such, plays an important role in cell growth, proliferation, and survival. Functional studies on lymphoblastoid cells with the PIK3R1 c.1906_1907insC mutation showed decreased phosphorylation of the downstream S6 target of the PI3K-AKT-mTOR pathway. Our findings show that PIK3R1 mutations are the major cause of SHORT syndrome and suggest that the molecular mechanism of disease might involve downregulation of the PI3K-AKT-mTOR pathway.     

Homozygous and Compound-Heterozygous Mutations in TGDS Cause Catel-Manzke Syndrome

Catel-Manzke syndrome is characterized by Pierre Robin sequence and a unique form of bilateral hyperphalangy causing a clinodactyly of the index finger. We describe the identification of homozygous and compound heterozygous mutations in TGDS in seven unrelated individuals with typical Catel-Manzke syndrome by exome sequencing. Six different TGDS mutations were detected: c.892A>G (p.Asn298Asp), c.270_271del (p.Lys91Asnfs^∗22), c.298G>T (p.Ala100Ser), c.294T>G (p.Phe98Leu), c.269A>G (p.Glu90Gly), and c.700T>C (p.Tyr234His), all predicted to be disease causing. By using haplotype reconstruction we showed that the mutation c.298G>T is probably a founder mutation. Due to the spectrum of the amino acid changes, we suggest that loss of function in TGDS is the underlying mechanism of Catel-Manzke syndrome. TGDS (dTDP-D-glucose 4,6-dehydrogenase) is a conserved protein belonging to the SDR family and probably plays a role in nucleotide sugar metabolism.     

FAD2 Gene Mutations Significantly Alter Fatty Acid Profiles in Cultivated Peanuts (Arachis hypogaea)

A panel of 55 peanut lines was analyzed for fatty acid composition with gas chromatography and also genotyped with SNP markers from the FAD2 genes by real-time PCR. Significant variation in fatty acid composition was identified, and the ratio of oleic acid to linoleic acid (O/L) ranged from 1.23 to 56.45. In terms of the FAD2 gene mutation, the assayed lines were classified into four genotypes: wild type (Ol(1)Ol(1)Ol(2)Ol(2)), single functional homozygous mutation on the A genome (ol(1)ol(1)Ol(2)Ol(2)), single functional homozygous mutation on the B genome (Ol(1)Ol(1)ol(2)ol(2)), and a double mutation on both A and B genomes (ol(1)ol(1)ol(2)ol(2)). Each genotype has a significantly different fatty acid profile. Both FAD2A and FAD2B are involved in the conversion of oleic acid to linoleic acid in peanuts. Overall, these results demonstrate the combined power of genetic analysis with biochemical analysis on peanut fatty acid research.