The genetic landscape and clinical implication of pediatric Moyamoya angiopathy in an international cohort.

Pediatric Moyamoya Angiopathy (MMA) is a progressive intracranial occlusive arteriopathy that represents a leading cause of transient ischemic attacks and strokes in childhood. Despite this, up to now no large, exclusively pediatric MMA cohort has been subjected to systematic genetic investigation. In this study, we performed molecular karyotyping, exome sequencing and automated structural assessment of missense variants on a series of 88 pediatric MMA patients and correlated genetic, angiographic and clinical (stroke burden) findings. The two largest subgroups in our cohort consisted of RNF213 and neurofibromatosis type 1 (NF1) patients. While deleterious RNF213 variants were associated with a severe MMA clinical course with early symptom onset, frequent posterior cerebral artery involvement and higher stroke rates in multiple territories, NF1 patients had a similar infarct burden compared to non-NF1 individuals and were often diagnosed incidentally during routine MRIs. Additionally, we found that MMA-associated RNF213 variants have lower predicted functional impact compared to those associated with aortic disease. We also raise the question of MMA as a feature of recurrent as well as rare chromosomal imbalances and further support the possible association of MMA with STAT3 deficiency. In conclusion, we provide a comprehensive characterization at the genetic and clinical level of a large exclusively pediatric MMA population. Due to the clinical differences found across genetic subgroups, we propose genetic testing for risk stratification as part of the routine assessment of pediatric MMA patients.

Bi-allelic variants in DOHH, catalyzing the last step of hypusine biosynthesis, are associated with a neurodevelopmental disorder.

Deoxyhypusine hydroxylase (DOHH) is the enzyme catalyzing the second step in the post-translational synthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] in the eukaryotic initiation factor 5A (eIF5A). Hypusine is formed exclusively in eIF5A by two sequential enzymatic steps catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusinated eIF5A is essential for translation and cell proliferation in eukaryotes, and all three genes encoding eIF5A, DHPS, and DOHH are highly conserved throughout eukaryotes. Pathogenic variants affecting either DHPS or EIF5A have been previously associated with neurodevelopmental disorders. Using trio exome sequencing, we identified rare bi-allelic pathogenic missense and truncating DOHH variants segregating with disease in five affected individuals from four unrelated families. The DOHH variants are associated with a neurodevelopmental phenotype that is similar to phenotypes caused by DHPS or EIF5A variants and includes global developmental delay, intellectual disability, facial dysmorphism, and microcephaly. A two-dimensional gel analyses revealed the accumulation of deoxyhypusine-containing eIF5A [eIF5A(Dhp)] and a reduction in the hypusinated eIF5A in fibroblasts derived from affected individuals, providing biochemical evidence for deficiency of DOHH activity in cells carrying the bi-allelic DOHH variants. Our data suggest that rare bi-allelic variants in DOHH result in reduced enzyme activity, limit the hypusination of eIF5A, and thereby lead to a neurodevelopmental disorder.

Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome.

BACKGROUND: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. RESULTS: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. CONCLUSIONS: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

Loss-of-function and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype.

PURPOSE: Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf-Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. METHODS: We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. RESULTS: The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2's folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. CONCLUSION: NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch-Steindl syndrome after the delineators of this phenotype.

Inborn errors of apolipoprotein A-I metabolism: implications for disease, research and development.

PURPOSE OF REVIEW: We review current knowledge regarding naturally occurring mutations in the human apolipoprotein A-I (APOA1) gene with a focus on their clinical complications as well as their exploitation for the elucidation of structure-function-(disease) relationships and therapy. RECENT FINDINGS: Bi-allelic loss-of-function mutations in APOA1 cause HDL deficiency and, in the majority of patients, premature atherosclerotic cardiovascular disease (ASCVD) and corneal opacities. Heterozygous HDL-cholesterol decreasing mutations in APOA1 were associated with increased risk of ASCVD in several but not all studies. Some missense mutations in APOA1 cause familial amyloidosis. Structure-function-reationships underlying the formation of amyloid as well as the manifestion of amyloidosis in specific tissues are better understood. Lessons may also be learnt from the progress in the treatment of amyloidoses induced by transthyretin variants. Infusion of reconstituted HDL (rHDL) containing apoA-I (Milano) did not cause regression of atherosclerosis in coronary arteries of patients with acute coronary syndrome. However, animal experiments indicate that rHDL with apoA-I (Milano) or apoA-I mimetic peptides may be useful for the treatment of heart failure of inflammatory bowel disease. SUMMARY: Specific mutations in APOA1 are the cause of premature ASCVD or familial amyloidosis. Synthetic mimetics of apoA-I (mutants) may be useful for the treatment of several diseases beyond ASCVD.

The hepatic WASH complex is required for efficient plasma LDL and HDL cholesterol clearance.

The evolutionary conserved Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex is one of the crucial multiprotein complexes that facilitates endosomal recycling of transmembrane proteins. Defects in WASH components have been associated with inherited developmental and neurological disorders in humans. Here, we show that hepatic ablation of the WASH component Washc1 in chow-fed mice increases plasma concentrations of cholesterol in both LDLs and HDLs, without affecting hepatic cholesterol content, hepatic cholesterol synthesis, biliary cholesterol excretion, or hepatic bile acid metabolism. Elevated plasma LDL cholesterol was related to reduced hepatocytic surface levels of the LDL receptor (LDLR) and the LDLR-related protein LRP1. Hepatic WASH ablation also reduced the surface levels of scavenger receptor class B type I and, concomitantly, selective uptake of HDL cholesterol into the liver. Furthermore, we found that WASHC1 deficiency increases LDLR proteolysis by the inducible degrader of LDLR, but does not affect proprotein convertase subtilisin/kexin type 9-mediated LDLR degradation. Remarkably, however, loss of hepatic WASHC1 may sensitize LDLR for proprotein convertase subtilisin/kexin type 9-induced degradation. Altogether, these findings identify the WASH complex as a regulator of LDL as well as HDL metabolism and provide in vivo evidence for endosomal trafficking of scavenger receptor class B type I in hepatocytes.

Complications and early mortality in percutaneous endoscopic gastrostomy placement in lombardy: A multicenter prospective cohort study.

BACKGROUND: Percutaneous endoscopic gastrostomy (PEG) is the most common endoscopic procedure used to provide nutritional support. AIM: To prospectively evaluate the mortality and complication incidences after PEG insertion or replacement. METHODS: All patients who underwent PEG insertion or replacement were included. Details on patient characteristics, ongoing therapies, comorbidities, and indication for PEG placement/replacement were collected, along with informed consent form signatures. Early and late (30-day) complications and mortality were assessed. RESULTS: 950 patients (47.1% male) were enrolled in 25 centers in Lombardy, a region of Northern Italy. Patient mean age was 73 years. 69.5% of patients had ASA status 3 or 4. First PEG placement was performed in 594 patients. Complication and mortality incidences were 4.8% and 5.2%, respectively. The most frequent complication was infection (50%), followed by bleeding (32.1%), tube dislodgment (14.3%), and buried bumper syndrome (3.6%). At multivariable analysis, age (OR 1.08 per 1-year increase, 95% CI, 1.0-1.16, p = 0.010) and BMI (OR 0.86 per 1-point increase, 95% CI, 0.77-0.96, p = 0.014) were factors associated with mortality. PEG replacement was carried out in 356 patients. Thirty-day mortality was 1.8%, while complications occurred in 1.7% of patients. CONCLUSIONS: Our data confirm that PEG placement is a safe procedure. Mortality was not related to the procedure itself, confirming that careful patient selection is warranted.

Endocytosis of lipoproteins.

During their metabolism, all lipoproteins undergo endocytosis, either to be degraded intracellularly, for example in hepatocytes or macrophages, or to be re-secreted, for example in the course of transcytosis by endothelial cells. Moreover, there are several examples of internalized lipoproteins sequestered intracellularly, possibly to exert intracellular functions, for example the cytolysis of trypanosoma. Endocytosis and the subsequent intracellular itinerary of lipoproteins hence are key areas for understanding the regulation of plasma lipid levels as well as the biological functions of lipoproteins. Indeed, the identification of the low-density lipoprotein (LDL)-receptor and the unraveling of its transcriptional regulation led to the elucidation of familial hypercholesterolemia as well as to the development of statins, the most successful therapeutics for lowering of cholesterol levels and risk of atherosclerotic cardiovascular diseases. Novel limiting factors of intracellular trafficking of LDL and the LDL receptor continue to be discovered and to provide drug targets such as PCSK9. Surprisingly, the receptors mediating endocytosis of high-density lipoproteins or lipoprotein(a) are still a matter of controversy or even new discovery. Finally, the receptors and mechanisms, which mediate the uptake of lipoproteins into non-degrading intracellular itineraries for re-secretion (transcytosis, retroendocytosis), storage, or execution of intracellular functions, are largely unknown.

Prevalence and causes of abnormal PSA recovery.

BACKGROUND: Prostate-specific antigen (PSA) test is of paramount importance as a diagnostic tool for the detection and monitoring of patients with prostate cancer. In the presence of interfering factors such as heterophilic antibodies or anti-PSA antibodies the PSA test can yield significantly falsified results. The prevalence of these factors is unknown. METHODS: We determined the recovery of PSA concentrations diluting patient samples with a standard serum of known PSA concentration. Based on the frequency distribution of recoveries in a pre-study on 268 samples, samples with recoveries <80% or >120% were defined as suspect, re-tested and further characterized to identify the cause of interference. RESULTS: A total of 1158 consecutive serum samples were analyzed. Four samples (0.3%) showed reproducibly disturbed recoveries of 10%, 68%, 166% and 4441%. In three samples heterophilic antibodies were identified as the probable cause, in the fourth anti-PSA-autoantibodies. The very low recovery caused by the latter interference was confirmed in serum, as well as heparin- and EDTA plasma of blood samples obtained 6 months later. Analysis by eight different immunoassays showed recoveries ranging between <10% and 80%. In a follow-up study of 212 random plasma samples we found seven samples with autoantibodies against PSA which however did not show any disturbed PSA recovery. CONCLUSIONS: About 0.3% of PSA determinations by the electrochemiluminescence assay (ECLIA) of Roche diagnostics are disturbed by heterophilic or anti-PSA autoantibodies. Although they are rare, these interferences can cause relevant misinterpretations of a PSA test result.

A human APOC3 missense variant and monoclonal antibody accelerate apoC-III clearance and lower triglyceride-rich lipoprotein levels.

Recent large-scale genetic sequencing efforts have identified rare coding variants in genes in the triglyceride-rich lipoprotein (TRL) clearance pathway that are protective against coronary heart disease (CHD), independently of LDL cholesterol (LDL-C) levels. Insight into the mechanisms of protection of these variants may facilitate the development of new therapies for lowering TRL levels. The gene APOC3 encodes apoC-III, a critical inhibitor of triglyceride (TG) lipolysis and remnant TRL clearance. Here we report a detailed interrogation of the mechanism of TRL lowering by the APOC3 Ala43Thr (A43T) variant, the only missense (rather than protein-truncating) variant in APOC3 reported to be TG lowering and protective against CHD. We found that both human APOC3 A43T heterozygotes and mice expressing human APOC3 A43T display markedly reduced circulating apoC-III levels. In mice, this reduction is due to impaired binding of A43T apoC-III to lipoproteins and accelerated renal catabolism of free apoC-III. Moreover, the reduced content of apoC-III in TRLs resulted in accelerated clearance of circulating TRLs. On the basis of this protective mechanism, we developed a monoclonal antibody targeting lipoprotein-bound human apoC-III that promotes circulating apoC-III clearance in mice expressing human APOC3 and enhances TRL catabolism in vivo. These data reveal the molecular mechanism by which a missense variant in APOC3 causes reduced circulating TG levels and, hence, protects from CHD. This protective mechanism has the potential to be exploited as a new therapeutic approach to reduce apoC-III levels and circulating TRL burden.

VEGF-A Regulates Cellular Localization of SR-BI as Well as Transendothelial Transport of HDL but Not LDL.

OBJECTIVE: Low- and high-density lipoproteins (LDL and HDL) must pass the endothelial layer to exert pro- and antiatherogenic activities, respectively, within the vascular wall. However, the rate-limiting factors that mediate transendothelial transport of lipoproteins are yet little known. Therefore, we performed a high-throughput screen with kinase drug inhibitors to identify modulators of transendothelial LDL and HDL transport. APPROACH AND RESULTS: Microscopy-based high-content screening was performed by incubating human aortic endothelial cells with 141 kinase-inhibiting drugs and fluorescent-labeled LDL or HDL. Inhibitors of vascular endothelial growth factor (VEGF) receptors (VEGFR) significantly decreased the uptake of HDL but not LDL. Silencing of VEGF receptor 2 significantly decreased cellular binding, association, and transendothelial transport of 125I-HDL but not 125I-LDL. RNA interference with VEGF receptor 1 or VEGF receptor 3 had no effect. Binding, uptake, and transport of HDL but not LDL were strongly reduced in the absence of VEGF-A from the cell culture medium and were restored by the addition of VEGF-A. The restoring effect of VEGF-A on endothelial binding, uptake, and transport of HDL was abrogated by pharmacological inhibition of phosphatidyl-inositol 3 kinase/protein kinase B or p38 mitogen-activated protein kinase, as well as silencing of scavenger receptor BI. Moreover, the presence of VEGF-A was found to be a prerequisite for the localization of scavenger receptor BI in the plasma membrane of endothelial cells. CONCLUSIONS: The identification of VEGF as a regulatory factor of transendothelial transport of HDL but not LDL supports the concept that the endothelium is a specific and, hence, druggable barrier for the entry of lipoproteins into the vascular wall.

Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease.

Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL) cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328 individuals with extremely high plasma HDL-C levels, we identified a homozygote for a loss-of-function variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells derived from induced pluripotent stem cells from the homozygous subject, and in mice. Large population-based studies revealed that subjects who are heterozygous carriers of the P376L variant have significantly increased levels of plasma HDL-C. P376L carriers have a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is statistically significant).

Reconstituted high-density lipoprotein can elevate plasma alanine aminotransferase by transient depletion of hepatic cholesterol: role of the phospholipid component.

Human apolipoprotein A-I preparations reconstituted with phospholipids (reconstituted high-density lipoprotein [HDL]) have been used in a large number of animal and human studies to investigate the physiological role of apolipoprotein A-I. Several of these studies observed that intravenous infusion of reconstituted HDL might cause transient elevations in plasma levels of hepatic enzymes. Here we describe the mechanism of this enzyme release. Observations from several animal models and in vitro studies suggest that the extent of hepatic transaminase release (alanine aminotransferase [ALT]) correlates with the movement of hepatic cholesterol into the blood after infusion. Both the amount of ALT release and cholesterol movement were dependent on the amount and type of phospholipid present in the reconstituted HDL. As cholesterol is known to dissolve readily in phospholipid, an HDL preparation was loaded with cholesterol before infusion into rats to assess the role of diffusion of cholesterol out of the liver and into the reconstituted HDL. Cholesterol-loaded HDL failed to withdraw cholesterol from tissues and subsequently failed to cause ALT release. To investigate further the role of cholesterol diffusion, we employed mice deficient in SR-BI, a transporter that facilitates spontaneous movement of cholesterol between cell membranes and HDL. These mice showed substantially lower movement of cholesterol into the blood and markedly lower ALT release. We conclude that initial depletion of hepatic cholesterol initiates transient ALT release in response to infusion of reconstituted HDL. This effect may be controlled by appropriate choice of the type and amount of phospholipid in reconstituted HDL. Copyright © 2015 John Wiley & Sons, Ltd.

Effects of miglustat treatment in a patient affected by an atypical form of Tangier disease.

BACKGROUND: Tangier disease (TD) is a rare autosomal recessive disorder, resulting from mutations in the ATP binding cassette transporter (ABCA1) gene. The deficiency of ABCA1 protein impairs high density lipoprotein (HDL) synthesis and cholesterol esters trafficking. CASE REPORT: A 58 year-old female, presenting with complex clinical signs (splenomegaly, dysarthria, dysphagia, ataxia, tongue enlargement, prurigo nodularis, legs lymphedema, pancytopenia and bone marrow foam cells), was misdiagnosed as Niemann-Pick C (NPC) and treated with miglustat (300 mg/day), normalizing neurological symptoms and improving skin lesions and legs lymphedema. Subsequently filipin-staining and molecular analysis for NPC genes were negative. Lipid profiling showed severe deficiency of HDL, 2 mg/dl (n.v. 45-65) and apoAI, 5.19 mg/dl (n.v. 110-170), suggesting TD as a probable diagnosis. Molecular analysis of ABCA1 gene showed the presence of a novel homozygous deletion (c.4464-486_4698 + 382 Del). Miglustat treatment was then interrupted with worsening of some neurological signs (memory defects, slowing of thought processes) and skin lesions. Treatment was restarted after 7 months with neurological normalization and improvement of skin involvement. CONCLUSIONS: These results suggest miglustat as a possible therapeutic approach in this untreatable disease. The mechanisms by which miglustat ameliorates at least some clinical manifestations of TD needs to be further investigated.

Association of low-frequency and rare coding-sequence variants with blood lipids and coronary heart disease in 56,000 whites and blacks.

Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the "Exome Array" to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121*], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited.

Novel mutations of ABCA1 transporter in patients with Tangier disease and familial HDL deficiency.

The objective of the study was the characterization of ABCA1 gene mutations in 10 patients with extremely low HDL-cholesterol. Five patients (aged 6 months to 76 years) presented with splenomegaly and thrombocytopenia suggesting the diagnosis of Tangier disease (TD). Three of them were homozygous for novel mutations either in intron (c.4465-34A>G) or in exons (c.4376delT and c.5449C>T), predicted to encode truncated proteins. One patient was compound heterozygous for a nucleotide insertion (c.1758_1759insG), resulting in a truncated protein and for a nucleotide substitution c.4799A>G, resulting in a missense mutation (p.H1600R). The last TD patient, found to be heterozygous for a known mutation (p.D1009Y), had a complete defect in ABCA1-mediated cholesterol efflux in fibroblasts, suggesting the presence of a second undetected mutant allele. Among the other patients, four were asymptomatic, but one, with multiple risk factors, had severe peripheral artery disease. Three of these patients were heterozygous for known mutations (p.R130K+p.N1800H, p.R1068C, p.N1800H), while two were carriers of novel mutations (c.1195-27G>A and c.396_397insA), predicted to encode truncated proteins. The pathogenic effect of the two intronic mutations (c. 1195-27G>A and c.4465-34A>G) was demonstrated by the analysis of the transcripts of splicing reporter mutant minigenes expressed in COS-1 cells. Both mutations activated an intronic acceptor splice site which resulted in a partial intron retention in mature mRNA with the production of truncated proteins. This study confirms the allelic heterogeneity of TD and suggests that the diagnosis of TD must be considered in patients with an unexplained splenomegaly, associated with thrombocytopenia and hypocholesterolemia.

Lysosomal lipase deficiency: molecular characterization of eleven patients with Wolman or cholesteryl ester storage disease.

Wolman Disease (WD) and cholesteryl ester storage disease (CESD) represent two distinct phenotypes of the same recessive disorder caused by the complete or partial deficiency of lysosomal acidic lipase (LAL), respectively. LAL, encoded by the LIPA gene, hydrolyzes cholesteryl esters derived from cell internalization of plasma lipoproteins. WD is a rapidly progressive and lethal disease characterized by intestinal malabsorption, hepatic and adrenal failure. CESD is characterized by hepatic fibrosis, hyperlipidemia and accelerated atherosclerosis. Aim of the study was the identification of LIPA mutations in three WD and eight CESD patients. The WD patients, all deceased before the first year of age, were homozygous for two novel mutations (c.299+1G>A and c.419G>A) or a mutation (c.796G>T) previously reported as compound heterozygosity in a CESD patient. The two mutations (c.419G>A and c.796G>T) resulting in truncated proteins (p.W140* and p.G266*) and the splicing mutation (c.229+1G>A) were associated with undetectable levels of LIPA mRNA in fibroblasts. All eight CESD patients carried the common mutation c.894G>A known to result not only in a major non-functional transcript with the skipping of exon 8 (p.S275_Q298del), but also in a minor normally spliced transcript producing 5-10% residual LAL activity. The c.894G>A mutation was found in homozygosity in four patients and, as compound heterozygosity, in association with a known (p.H295Y and p.G342R) or a novel (p.W140*) mutation in four other CESD patients. Segregation analysis performed in all patients harboring c.895G>A showed its occurrence on the same haplotype suggesting a common founder ancestor. The other WD and CESD mutations were associated with different haplotypes.