A common Lithuanian mutation causing familial hypercholesterolemia in Ashkenazi Jews.

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low-density-lipoprotein (LDL) receptor. Here we characterize an LDL-receptor founder mutation that is associated with a distinct LDL-receptor haplotype and is responsible for FH in 35% of 71 Jewish-Ashkenazi FH families in Israel. Sixty four percent (16/25) of the Ashkenazi patients who carry this mutant allele were of Lithuanian origin. The mutation was not found in 47 non-Ashkenazi FH families. This mutation was prevalent (8/10 FH cases) in the Jewish community in South Africa, which originated mainly from Lithuania. The mutation, a 3-bp in-frame deletion that would result in the elimination of Gly197, has been previously designated FH-Piscataway. PCR amplification of a DNA fragment that includes the mutation in heterozygous individuals results in the formation of a heteroduplex that can be demonstrated by PAGE and used for molecular diagnosis.     

On the mechanism of accumulation of cholestanol in the brain of mice with a disruption of sterol 27-hydroxylase

The rare disease cerebrotendinous xanthomatosis (CTX) is due to a lack of sterol 27-hydroxylase (CYP27A1) and is characterized by cholestanol-containing xanthomas in brain and tendons. Mice with the same defect do not develop xanthomas. The driving force in the development of the xanthomas is likely to be conversion of a bile acid precursor into cholestanol. The mechanism behind the xanthomas in the brain has not been clarified. We demonstrate here that female cyp27a1^−/− mice have an increase of cholestanol of about 2.5- fold in plasma, 6-fold in tendons, and 12-fold in brain. Treatment of cyp27a1^−/− mice with 0.05% cholic acid normalized the cholestanol levels in tendons and plasma and reduced the content in the brain. The above changes occurred in parallel with changes in plasma levels of 7α-hydroxy-4-cholesten-3-one, a precursor both to bile acids and cholestanol. Injection of a cyp27a1^−/− mouse with ²H₇-labeled 7α-hydroxy-4-cholesten-3-one resulted in a significant incorporation of ²H₇-cholestanol in the brain. The results are consistent with a concentration-dependent flux of 7α-hydroxy-4-cholesten-3-one across the blood-brain barrier in cyp27a1^−/− mice and subsequent formation of cholestanol. It is suggested that the same mechanism is responsible for accumulation of cholestanol in the brain of patients with CTX.     

Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel syndrome

Meckel syndrome (MKS) is a rare autosomal recessive lethal condition characterized by central nervous system malformations, polydactyly, multicystic kidney dysplasia, and ductal changes of the liver. Three loci have been mapped (MKS1–MKS3), and two genes have been identified (MKS1/FLJ20345 and MKS3/TMEM67), whereas the gene at the MKS2 locus remains unknown. To identify new MKS loci, a genomewide linkage scan was performed using 10-cM–resolution microsatellite markers in eight families. The highest heterogeneity LOD score was obtained for chromosome 12, in an interval containing CEP290, a gene recently identified as causative of Joubert syndrome (JS) and isolated Leber congenital amaurosis. In view of our recent findings of allelism, at the MKS3 locus, between these two disorders, CEP290 was considered a candidate, and homozygous or compound heterozygous truncating mutations were identified in four families. Sequencing of additional cases identified CEP290 mutations in two fetuses with MKS and in four families presenting a cerebro-reno-digital syndrome, with a phenotype overlapping MKS and JS, further demonstrating that MKS and JS can be variable expressions of the same ciliopathy. These data identify a fourth locus for MKS (MKS4) and the CEP290 gene as responsible for MKS.     

Parent-of-Origin Effects of the APOB Gene on Adiposity in Young Adults

Loci identified in genome-wide association studies (GWAS) of cardio-metabolic traits account for a small proportion of the traits'' heritability. To date, most association studies have not considered parent-of-origin effects (POEs). Here we report investigation of POEs on adiposity and glycemic traits in young adults. The Jerusalem Perinatal Family Follow-Up Study (JPS), comprising 1250 young adults and their mothers was used for discovery. Focusing on 18 genes identified by previous GWAS as associated with cardio-metabolic traits, we used linear regression to examine the associations of maternally- and paternally-derived offspring minor alleles with body mass index (BMI), waist circumference (WC), fasting glucose and insulin. We replicated and meta-analyzed JPS findings in individuals of European ancestry aged ≤50 belonging to pedigrees from the Framingham Heart Study, Family Heart Study and Erasmus Rucphen Family study (total N≅4800). We considered p<2.7x10^-4 statistically significant to account for multiple testing. We identified a common coding variant in the 4^th exon of APOB (rs1367117) with a significant maternally-derived effect on BMI (β = 0.8; 95%CI:0.4,1.1; p = 3.1x10^-5) and WC (β = 2.7; 95%CI:1.7,3.7; p = 2.1x10^-7). The corresponding paternally-derived effects were non-significant (p>0.6). Suggestive maternally-derived associations of rs1367117 were observed with fasting glucose (β = 0.9; 95%CI:0.3,1.5; p = 4.0x10^-3) and insulin (ln-transformed, β = 0.06; 95%CI:0.03,0.1; p = 7.4x10^-4). Bioinformatic annotation for rs1367117 revealed a variety of regulatory functions in this region in liver and adipose tissues and a 50% methylation pattern in liver only, consistent with allelic-specific methylation, which may indicate tissue-specific POE. Our findings demonstrate a maternal-specific association between a common APOB variant and adiposity, an association that was not previously detected in GWAS. These results provide evidence for the role of regulatory mechanisms, POEs specifically, in adiposity. In addition this study highlights the benefit of utilizing family studies for deciphering the genetic architecture of complex traits.     

Recent origin and spread of a common Lithuanian mutation, G197del LDLR, causing familial hypercholesterolemia: positive selection is not always necessary to account for disease incidence among Ashkenazi Jews

G197del is the most prevalent LDL receptor (LDLR) mutation causing familial hypercholesterolemia (FH) in Ashkenazi Jew (AJ) individuals. The purpose of this study was to determine the origin, age, and population distribution of G197del, as well as to explore environmental and genetic effects on disease expression. Index cases from Israel (n=46), South Africa (n=24), Russia (n=7), The Netherlands (n=1), and the United States (n=1) were enlisted. All trace their ancestry to Lithuania. A highly conserved haplotype (D19S221:104-D19S865:208-D19S413:74) was identified in G197del chromosomes, suggesting the occurrence of a common founder. When two methods were used for analysis of linkage disequilibrium (LD) between flanking polymorphic markers and the disease locus and for the study of the decay of LD over time, the estimated age of the deletion was found to be 20 +/- 7 generations (the 95% confidence interval is 15-26 generations), so that the most recent common ancestor of the mutation-bearing chromosomes would date to the 14th century. This corresponds with the founding of the Jewish community of Lithuania (1338 a.d.), as well as with the great demographic expansion of AJ individuals in eastern Europe, which followed this settlement. The penetrance of mutation-linked severe hypercholesterolemia is high (94% of heterozygotes have a baseline concentration of LDL cholesterol (LDL-C) that is >160 mg/dl), and no significant differences in the mean baseline lipid level of G197del carriers from different countries were found. Polymorphisms of apolipoprotein E and of scavenger-receptor class B type I were observed to have minor effects on the plasma lipid profile. With respect to determinative genetic influences on the biochemical phenotype, there is no evidence that could support the possibility of a selective evolutionary metabolic advantage. Therefore, the founder effect in a rapidly expanding population from a limited number of families remains a simple, parsimonious hypothesis explaining the spread of G197del-LDLR-linked FH in AJ individuals.     

Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.     

Complete penetrance of Creutzfeldt-Jakob disease in Libyan Jews carrying the E200K mutation in the prion protein gene.

BACKGROUND: Creutzfeldt-Jakob disease (CJD) is a prion disease which is manifest as a sporadic, inherited, and transmissible neurodegenerative disorder. The mean age at onset of CJD is approximately 60 years, and as such, many people destined to succumb undoubtedly die of other illnesses first. The delayed onset of CJD has complicated the analysis of inherited forms of the illness and led to the suggestion that mutations in the prion protein (PrP) gene are necessary but not sufficient for prion disease despite genetic linkage; indeed, an environmental factor such as a ubiquitous virus has been proposed as a second necessary factor. MATERIALS AND METHODS: To examine what appeared to be incomplete penetrance, we applied a life-table analysis to clinical and pedigree data from a cluster population of Libyan Jews in which the E200K mutation is prevalent. The study population included 42 affected and 44 unaffected members of 13 Libyan Jewish families, all of whom possessed the E200K mutation. RESULTS: The calculated value using life table analysis is 0.77 at age 70 which increases to 0.89 if a mutation carrier survives to age 80 and 0.96 if age 80 is surpassed. CONCLUSIONS: These data argue that the E200K mutation alone is sufficient to cause prion disease and does so in an age-dependent manner.     

A nonsense mutation in the LDL receptor gene leads to familial hypercholesterolemia in the Druze sect.

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the LDL receptor gene. Here we characterize an LDL receptor mutation that is associated with a distinct haplotype and causes FH in the Druze, a small Middle Eastern Islamic sect with a high degree of inbreeding. The mutation was found in FH families from two distinct Druze villages from the Golan Heights (northern Israel). It was not found neither in another Druze FH family residing in a different geographical area nor in eight Arab and four Jewish FH heterozygote index cases whose hypercholesterolemia cosegregates with an identical LDL receptor gene haplotype. The mutation, a single-base substitution, results in a termination codon in exon 4 of the LDL receptor gene that encodes for the fourth repeat of the binding domain of the mature receptor. It can be diagnosed by allele-specific oligonucleotide hybridization of PCR-amplified DNA from FH patients.     

Heterozygous mutations in SIX3 and SHH are associated with schizencephaly and further expand the clinical spectrum of holoprosencephaly

Schizencephaly (SCH) is a clinically and etiologically heterogeneous cerebral malformation presenting as unilateral or bilateral hemispheric cleft with direct connection between the inner and outer liquor spaces. The SCH cleft is usually lined by gray matter, which appears polymicrogyric implying an associated impairment of neuronal migration. The majority of SCH patients are sporadic, but familial SCH has been described. An initial report of heterozygous mutations in the homeobox gene EMX2 could not be confirmed in 52 patients investigated in this study in agreement with two independent SCH patient cohorts published previously. SCH frequently occurs with additional cerebral malformations like hypoplasia or aplasia of the septum pellucidum or optic nerve, suggesting the involvement of genes important for the establishment of midline forebrain structures. We therefore considered holoprosencephaly (HPE)-associated genes as potential SCH candidates and report for the first time heterozygous mutations in SIX3 and SHH in a total of three unrelated patients and one fetus with SCH; one of them without obvious associated malformations of midline forebrain structures. Three of these mutations have previously been reported in independent patients with HPE. SIX3 acts directly upstream of SHH, and the SHH pathway is a key regulator of ventral forebrain patterning. Our data indicate that in a subset of patients SCH may develop as one aspect of a more complex malformation of the ventral forebrain, directly result from mutations in the SHH pathway and hence be considered as yet another feature of the broad phenotypic spectrum of holoprosencephaly.     

Fatal prion disease in a mouse model of genetic E200K Creutzfeldt-Jakob disease

Genetic prion diseases are late onset fatal neurodegenerative disorders linked to pathogenic mutations in the prion protein-encoding gene, PRNP. The most prevalent of these is the substitution of Glutamate for Lysine at codon 200 (E200K), causing genetic Creutzfeldt-Jakob disease (gCJD) in several clusters, including Jews of Libyan origin. Investigating the pathogenesis of genetic CJD, as well as developing prophylactic treatments for young asymptomatic carriers of this and other PrP mutations, may well depend upon the availability of appropriate animal models in which long term treatments can be evaluated for efficacy and toxicity. Here we present the first effective mouse model for E200KCJD, which expresses chimeric mouse/human (TgMHu2M) E199KPrP on both a null and a wt PrP background, as is the case for heterozygous patients and carriers. Mice from both lines suffered from distinct neurological symptoms as early as 5-6 month of age and deteriorated to death several months thereafter. Histopathological examination of the brain and spinal cord revealed early gliosis and age-related intraneuronal deposition of disease-associated PrP similarly to human E200K gCJD. Concomitantly we detected aggregated, proteinase K resistant, truncated and oxidized PrP forms on immunoblots. Inoculation of brain extracts from TgMHu2ME199K mice readily induced, the first time for any mutant prion transgenic model, a distinct fatal prion disease in wt mice. We believe that these mice may serve as an ideal platform for the investigation of the pathogenesis of genetic prion disease and thus for the monitoring of anti-prion treatments.