Torunn Fiskerstrand

Germline mutations in WTX cause a sclerosing skeletal dysplasia but do not predispose to tumorigenesis

Abnormalities in WNT signaling are implicated in a broad range of developmental anomalies and also in tumorigenesis. Here we demonstrate that germline mutations in WTX (FAM123B), a gene that encodes a repressor of canonical WNT signaling, cause an X-linked sclerosing bone dysplasia, osteopathia striata congenita with cranial sclerosis (OSCS; MIM300373). This condition is typically characterized by increased bone density and craniofacial malformations in females and lethality in males. The mouse homolog of WTX is expressed in the fetal skeleton, and alternative splicing implicates plasma membrane localization of WTX as a factor associated with survival in males with OSCS. WTX has also been shown to be somatically inactivated in 11–29% of cases of Wilms tumor. Despite being germline for such mutations, individuals with OSCS are not predisposed to tumor development. The observed phenotypic discordance dependent upon whether a mutation is germline or occurs somatically suggests the existence of temporal or spatial constraints on the action of WTX during tumorigenesis.

Ciliopathies with Skeletal Anomalies and Renal Insufficiency due to Mutations in the IFT-A Gene WDR19

A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.

Mutations in ABHD12 Cause the Neurodegenerative Disease PHARC: An Inborn Error of Endocannabinoid Metabolism

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a neurodegenerative disease marked by early-onset cataract and hearing loss, retinitis pigmentosa, and involvement of both the central and peripheral nervous systems, including demyelinating sensorimotor polyneuropathy and cerebellar ataxia. Previously, we mapped this Refsum-like disorder to a 16 Mb region on chromosome 20. Here we report that mutations in the ABHD12 gene cause PHARC disease and we describe the clinical manifestations in a total of 19 patients from four different countries. The ABHD12 enzyme was recently shown to hydrolyze 2-arachidonoyl glycerol (2-AG), the main endocannabinoid lipid transmitter that acts on cannabinoid receptors CB1 and CB2. Our data therefore represent an example of an inherited disorder related to endocannabinoid metabolism. The endocannabinoid system is involved in a wide range of physiological processes including neurotransmission, mood, appetite, pain appreciation, addiction behavior, and inflammation, and several potential drugs targeting these pathways are in development for clinical applications. Our findings show that ABHD12 performs essential functions in both the central and peripheral nervous systems and the eye. Any future drug-mediated interference with this enzyme should consider the potential risk of long-term adverse effects.

Familial diarrhea syndrome caused by an activating GUCY2C mutation.

BACKGROUND Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS We identified a heterozygous missense mutation (c.2519G→T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohns disease is warranted. (Funded by Helse Vest [Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.).

Assessment of homocysteine status

Plasma total homocysteine (tHcy) determination is used in the diagnosis of homocystinuria, in cobalamin and folate deficiency and in cardiovascular risk assessment. However, determination of tHcy includes many pitfalls which complicate the assessment of homocysteine status. In the present article, we review basic knowledge for a rational use of plasma tHcy in diagnostic as well as scientific work. The subjects dealt with are procedures for sample handling and processing, the principles of tHcy analyses, and genetic and acquired determinants of the plasma tHcy concentration.

Dominant Mutations in the Autoimmune Regulator AIRE Are Associated with Common Organ-Specific Autoimmune Diseases.

The autoimmune regulator (AIRE) gene is crucial for establishing central immunological tolerance and preventing autoimmunity. Mutations in AIRE cause a rare autosomal-recessive disease, autoimmune polyendocrine syndrome type 1 (APS-1), distinguished by multi-organ autoimmunity. We have identified multiple cases and families with mono-allelic mutations in the first plant homeodomain (PHD1) zinc finger of AIRE that followed dominant inheritance, typically characterized by later onset, milder phenotypes, and reduced penetrance compared to classical APS-1. These missense PHD1 mutations suppressed gene expression driven by wild-type AIRE in a dominant-negative manner, unlike CARD or truncated AIRE mutants that lacked such dominant capacity. Exome array analysis revealed that the PHD1 dominant mutants were found with relatively high frequency (>0.0008) in mixed populations. Our results provide insight into the molecular action of AIRE and demonstrate that disease-causing mutations in the AIRE locus are more common than previously appreciated and cause more variable autoimmune phenotypes.

Frontometaphyseal dysplasia: mutations in FLNA and phenotypic diversity.

Frontometaphyseal dysplasia is an X‐linked trait primarily characterized by a skeletal dysplasia comprising hyperostosis of the skull and modeling anomalies of the tubular bones. Extraskeletal features include tracheobronchial, cardiac, and urological malformations. A proportion of individuals have missense mutations or small deletions in the X‐linked gene, FLNA. We report here our experience with comprehensive screening of the FLNA gene in a group of 23 unrelated probands (11 familial instances, 12 simplex cases; total affected individuals 32) with FMD. We found missense mutations leading to substitutions in the actin‐binding domain and within filamin repeats 9, 10, 14, 16, 22, and 23 of filamin A in 13/23 (57%) of individuals in this cohort. Some mutations present with a male phenotype that is characterized by a severe skeletal dysplasia, cardiac, and genitourinary malformations that leads to perinatal death. Although no phenotypic feature consistently discriminates between females with FMD who are heterozygous for FLNA mutations and those in whom no FLNA mutation can be identified, there is a difference in the degree of skewing of X‐inactivation between these two groups. This observation suggests that locus heterogeneity may exist for this disorder.

Hyperhomocysteinemia in terms of steady-state kinetics.

Abstract The plasma level of homocysteine (Hcy) and its oxidized products, i.e., plasma total Hcy (tHcy), is a function of the influx rate of Hcy to plasma and the plasma tHcy clearance. In vitro experiments show that proliferating cells usually export more Hcy than stationary cells and that the Hcy export increases in response to high methionine, low folate or low cobalamin level, and to agents interfering with Hcy remethylation. Comparison between various cell types suggests that hepatocytes have a unique ability to increase the Hcy export in response to extracellular methionine, probably due to its capacity to form adenosylmethionine. Some but not all cell types have an ability to use extracellular Hcy as a methionine source. Clearance studies in healthy subjects indicate that about 1.2 mmol Hcy is supplied from the cells to plasma per 24 h, which is only about 5–10% of total Hcy formed. Comparison of area under the curves after administration of Hcy and methionine shows that about 10% of the methionine administered is released to plasma as Hcy. Notably, only a few percent of Hcy from plasma is excreted unchanged in the urine, and this shows that most tHcy in plasma is metabolized. Folate or cobalamin deficient patients have normal plasma tHcy clearance, which suggests that their elevated tHcy level is due to increased Hcy export from tissues into the plasma compartment. In contrast, the hyperhomocysteinemia in renal failure is accounted for by a marked reduction in tHcy clearance, suggesting an important role of kidney in elimination of Hcy from plasma.

Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG)

Osteoporosis-pseudoglioma sydrome (OPPG) is an autosomal recessive disorder with early-onset severe osteoporosis and blindness, caused by biallelic loss-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Heterozygous carriers exhibit a milder bone phenotype. Only a few splice mutations in LRP5 have been published. We present clinical and genetic data for four patients with novel LRP5 mutations, three of which affect splicing. Patients were evaluated clinically and by radiography and bone densitometry. Genetic screening of LRP5 was performed on the basis of the clinical diagnosis of OPPG. Splice aberrances were confirmed by cDNA sequencing or exon trapping. The effect of one splice mutation on LRP5 protein function was studied. A novel splice-site mutation c.1584+4A>T abolished the donor splice site of exon 7 and activated a cryptic splice site, which led to an in-frame insertion of 21 amino acids (p.E528_V529ins21). Functional studies revealed severely impaired signal transduction presumably caused by defective intracellular transport of the mutated receptor. Exon trapping was used on two samples to confirm that splice-site mutations c.4112-2A>G and c.1015+1G>T caused splicing-out of exons 20 and 5, respectively. One patient carried a homozygous deletion of exon 4 causing the loss of exons 4 and 5, as demonstrated by cDNA analysis. Our results broaden the spectrum of mutations in LRP5 and provide the first functional data on splice aberrations.

A novel ADAMTSL4 mutation in autosomal recessive ectopia lentis et pupillae.

PURPOSE To examine the ocular malformations and identify the molecular genetic basis for autosomal recessive ectopia lentis et pupillae in five Norwegian families. METHODS Ten affected persons and 11 first-degree relatives of five Norwegian families underwent ophthalmic and general medical examination. Molecular genetic studies included homozygosity mapping with SNP markers, DNA sequencing, and RT-PCR analysis. RESULTS Ocular signs in affected persons were increased median corneal thickness and astigmatism, angle malformation with prominent iris processes, displacement of the pupil and lens, lens coloboma, spherophakia, loss of zonular threads, early cataract development, glaucoma, and retinal detachment. No cardiac or metabolic abnormalities known to be associated with ectopia lentis were detected. Affected persons shared a 0.67 cM region of homozygosity on chromosome 1. DNA sequencing revealed a novel mutation in ADAMTSL4, c.767_786del20. This deletion of 20 base pairs (bp) results in a frameshift and an introduction of a stop codon 113 bp downstream, predicting a C-terminal truncation of the ADAMTSL4 protein (p.Gln256ProfsX38). Expression of truncated ADAMTSL4 mRNA was confirmed by RT-PCR analysis. Three of 190 local blood donors were carriers of this mutation. CONCLUSIONS Ectopia lentis et pupillae is associated with a number of malformations primarily in the anterior segment of the eye. The causative mutation, which is the first to be described in ectopia lentis et pupillae, disrupts the same gene function previously shown to cause isolated ectopia lentis. The mutation is ancient and may, therefore, be spread to a much larger population than the investigated one.