Human Mendelian pain disorders: a key to discovery and validation of novel analgesics.

We have utilized a novel application of human genetics, illuminating the important role that rare genetic disorders can play in the development of novel drugs that may be of relevance for the treatment of both rare and common diseases. By studying a very rare Mendelian disorder of absent pain perception, congenital indifference to pain, we have defined Nav1.7 (endocded by SCN9A) as a critical and novel target for analgesic development. Strong human validation has emerged with SCN9A gain-of-function mutations causing inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder, both Mendelian disorder of spontaneous or easily evoked pain. Furthermore, variations in the Nav1.7 channel also modulate pain perception in healthy subjects as well as in painful conditions such as osteoarthritis and Parkinson disease. On the basis of this, we have developed a novel compound (XEN402) that exhibits potent, voltage-dependent block of Nav1.7. In a small pilot study, we showed that XEN402 blocks Nav1.7 mediated pain associated with IEM thereby demonstrating the use of rare genetic disorders with mutant target channels as a novel approach to rapid proof-of-concept. Our approach underscores the critical role that human genetics can play by illuminating novel and critical pathways pertinent for drug discovery.

Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations.

Congenital indifference to pain (CIP) is a rare condition in which patients have severely impaired pain perception, but are otherwise essentially normal. We identified and collected DNA from individuals from nine families of seven different nationalities in which the affected individuals meet the diagnostic criteria for CIP. Using homozygosity mapping and haplotype sharing methods, we narrowed the CIP locus to chromosome 2q24-q31, a region known to contain a cluster of voltage-gated sodium channel genes. From these prioritized candidate sodium channels, we identified 10 mutations in the SCN9A gene encoding the sodium channel protein Nav1.7. The mutations completely co-segregated with the disease phenotype, and nine of these SCN9A mutations resulted in truncation and loss-of-function of the Nav1.7 channel. These genetic data further support the evidence that Nav1.7 plays an essential role in mediating pain in humans, and that SCN9A mutations identified in multiple different populations underlie CIP.

Allelic variation in the promoter region of the LDL receptor gene: analysis of an African-specific variant in the FP2 cis-acting regulatory element.

DNA samples of 2303 individuals from nine different population groups were screened for variant -175g-->t in the promoter region of the low-density lipoprotein receptor (LDLR) gene. The -175g-->t variant detected at carrier frequencies of 3-10% in different African population groups was absent in the Caucasian and Asian (Chinese) individuals studied. In contrast to previous findings in Black South Africans where this polymorphism predominated in patients with familial hypercholesterolaemia (FH), it occurred at a significantly lower frequency in hypercholesterolaemics from the recently admixed Coloured population of South Africa compared with population-matched controls (P<0.0001). Haplotype and mutation analysis excluded the likelihood that this finding is due to association with a specific disease-related mutation in FH patients, although reversal of the positive association with FH observed in the Black population may, at least in part, be due to admixture linkage disequilibrium. Transient transfection studies in HepG2 cells demonstrated that the -175t allele is associated with a non-significant decrease ( approximately 7%) of LDLR transcription in the absence of sterols. The data presented in this study raise the possibility that the -175g-->t polymorphism may have subtle effects that become clinically important within certain genetic and/or environmental contexts.

Common mutations in the lipoprotein lipase gene (LPL): effects on HDL-cholesterol levels in a Chinese Canadian population.

BACKGROUND: favorable lipid profiles including low total serum cholesterol (TC), TC/HDL-cholesterol (HDL-C) ratio and elevated HDL-C levels have been previously reported in Chinese living in China. More recent data, however, suggests a changing trend toward decreased HDL-C and increased TC and LDL cholesterol (LDL-C) in Chinese populations. Environmental factors likely contribute, in part, to these findings. However, genetic factors contributing to lipoprotein metabolism may also play a role in determining the lipid/lipoprotein phenotype observed in Chinese populations. Lipoprotein lipase (LPL) mutations have been associated with altered HDL-C concentrations in Caucasians but have not yet been studied in a large population of Chinese descent. METHODS: 1577 Chinese Canadians of Cantonese descent were recruited for a cardiovascular risk factor study. The frequency and effect of three LPL gene polymorphisms [Asp9Asn (D9N, n=374), Asn291Ser (N291S, n=321) and Ser447-Ter (S447X, n=403)] on serum HDL-C concentrations was assessed. All the three polymorphisms have been shown to alter HDL-C levels in different Caucasian populations. RESULTS: lower TC, LDL-C, and TG and higher HDL-C were observed in both male and female Chinese Canadian subjects compared to other population samples. The D9N and N291S LPL polymorphisms were identified in 1/374 (0.3%) and 5/321 (1.6%) subjects, respectively. Carrier frequency of the S447X mutation was (102/403) 25.3%. This S447X polymorphism was observed with higher frequency in males with HDL-C levels in the highest tertile compared with those in the lowest HDL-C tertile (carrier frequencies 37.3 vs. 19.4%) (P=0.046). CONCLUSION: in this cohort of Chinese Canadians, the serum lipid profiles were more favorable than what has been reported for Caucasian Canadians. A favorable spectrum of polymorphisms in the LPL gene may mitigate the adverse effects of western lifestyle on plasma lipoproteins in this cohort of Cantonese Canadians.

The factor structure for positive and negative symptoms in South African Xhosa patients with schizophrenia.

Most studies investigating the symptom dimensions of schizophrenia utilising the Scale for the Assessment of Negative Symptoms (SANS) and the Scale for the Assessment of Positive Symptoms (SAPS) favour a three factor model. This study sought to investigate the factor structure of both the global and individual items of the SANS and SAPS in a large sample of South African Xhosa patients with schizophrenia. A total of 422 subjects participated. Both principal components and factor analytical procedures were applied. For the global items, a two-factor solution representing positive and negative symptoms accounted for 59.9% of the variance. Alternatively, the three-dimensional model of negative, psychotic and disorganisation factors was supported by a five-factor solution if the more heterogeneous items of attention and alogia were ignored. Analysis of the individual items yielded a five-factor solution with the negative symptoms splitting into diminished expression and disordered relating, and the positive symptoms separating into factors for psychosis, thought disorder and bizarre behaviour. Our findings are very similar to those from other parts of the world, providing evidence that the factor structure for the symptoms of schizophrenia is relatively resistant to cultural influences. This is particularly true for negative symptoms.

A frequent mutation in the lipoprotein lipase gene (D9N) deteriorates the biochemical and clinical phenotype of familial hypercholesterolemia.

The D9N substitution is a common mutation in the lipoprotein lipase (LPL) gene. This mutation has been associated with reduced levels of HDL cholesterol and elevated triglycerides (TG) in a wide variety of patients. We investigated the influence of this D9N mutation on lipid and lipoprotein levels and risk for cardiovascular disease (CVD) in patients with familial hypercholesterolemia (FH). A total of 2091 FH heterozygotes, all of Dutch extraction, were screened for the D9N mutation using standard polymerase chain reaction techniques, followed by specific enzyme digestion. A total of 94 FH subjects carried the D9N mutation at a carrier frequency of 4.5%. Carriers of other common LPL mutations, such as the N291S and the S447X were excluded. Clinical data on 80 FH individuals carrying the D9N were available and were compared with a FH control group matched for age, sex, and body mass index (n=203). Analysis revealed significantly higher TG (P=0.01) and lower HDL-cholesterol levels (P=0.002). Dyslipidemia was more pronounced in D9N carriers with higher body mass index. Moreover, FH patients carrying this common LPL mutation were at higher risk for CVD, (odds ratio=2.8; 95% CI, 1.43 to 5.32; P=0.002). The common D9N LPL mutation leads to increased TG and decreased HDL plasma levels in patients with FH. These effects are most apparent in those FH heterozygotes with an increased body mass index. Furthermore, this mutation, present in 4.5% of Dutch FH heterozygotes, leads to increased risk for CVD.

Mutations in the ABC1 gene in familial HDL deficiency with defective cholesterol efflux.

BACKGROUND: A low concentration of HDL cholesterol is the most common lipoprotein abnormality in patients with premature atherosclerosis. We have shown that Tangier disease, a rare and severe form of HDL deficiency characterised by a biochemical defect in cellular cholesterol efflux, is caused by mutations in the ATP-binding-cassette (ABC1) gene. This gene codes for the cholesterol-efflux regulatory protein (CERP). We investigated the presence of mutations in this gene in patients with familial HDL deficiency. METHODS: Three French-Canadian families and one Dutch family with familial HDL deficiency were studied. Fibroblasts from the proband of each family were defective in cellular cholesterol efflux. Genomic DNA of each proband was used for mutation detection with primers flanking each exon of the ABC1 gene, and for sequencing of the entire coding region of the gene. PCR and restriction-fragment length polymorphism assays specific to each mutation were used to investigate segregation of the mutation in each family, and to test for absence of the mutation in DNA from normal controls. FINDINGS: A different mutation was detected in ABC1 in each family studied. Each mutation either created a stop codon predicted to result in truncation of CERP, or altered a conserved aminoacid residue. Each mutation segregated with low concentrations of HDL-cholesterol in the family, and was not observed in more than 500 control chromosomes tested. INTERPRETATION: These data show that mutations in ABC1 are the major cause of familial HDL deficiency associated with defective cholesterol efflux, and that CERP has an essential role in the formation of HDL. Our findings highlight the potential of modulation of ABC1 as a new route for increasing HDL concentrations.

A common truncation variant of lipoprotein lipase (Ser447X) confers protection against coronary heart disease: the Framingham Offspring Study.

Genetic variation at the lipoprotein lipase (LPL) locus has been shown to influence plasma lipids and to modulate risk of coronary heart disease (CHD). Recently, we found that the most frequent variant at this locus, involving a C-terminal truncation of two amino acids (Ser447X), was associated with both higher LPL activity and high density lipoprotein cholesterol (HDL-C) in patients with CHD. However, the impact of this S447X variant on lipids and CHD in the general population was hitherto unknown. We, therefore, analyzed a total of 1114 men and 1144 women randomly ascertained from the Framingham Offspring Study (FOS) for the presence of this LPL variant. Carrier frequency of the S447X allele was 17%, and in men carrier status was associated with higher total cholesterol (delta = 6.2 mg/dl, p = 0.03). higher HDL-C (delta = 2.3 mg/dl, p = 0.01), and lower triglyceride (TG) levels (delta = -19.4 mg/dl, p = 0.02). Moreover, in men, the S447X allele conferred significant protection against CHD (odds ratio: 0.43; p = 0.04). These effects on lipids and CHD were not seen in women. Our study represents the first report on the impact of this mutation on CHD in men from the general population, and we conclude, therefore, that the S447X variant may confer significant protection against high TG levels, low HDL-C, and premature CHD in these subjects.

Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.

Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette transporter (ABC1). Familial HDL deficiency (FHA) is a more frequent cause of low HDL levels. On the basis of independent linkage and meiotic recombinants, we localized the FHA locus to the same genomic region as the TD locus. Mutations in ABC1 were detected in both TD and FHA, indicating that TD and FHA are allelic. This indicates that the protein encoded by ABC1 is a key gatekeeper influencing intracellular cholesterol transport, hence we have named it cholesterol efflux regulatory protein (CERP).

Two common mutations (D9N, N291S) in lipoprotein lipase: a cumulative analysis of their influence on plasma lipids and lipoproteins in men and women.

We assessed the effect of two common mutations in the lipoprotein lipase gene (LPL), D9N and N291S, which have been shown to modulate plasma lipids in a wide spectrum of patients. A total of 1114 men and 1 144 women from the Framingham Offspring Study (FOS) were analyzed for these two LPL variants. Subsequently, the association with fasting plasma lipids and risk of coronary artery disease (CHD) was determined. We extended our study by calculating weighed means of lipids and lipoproteins in carriers and non-carriers for these LPL mutations in patients with genetic dyslipidemias, CHD patients and healthy controls. In the FOS sample, the D9N and N291S alleles were associated with lower high-density lipoprotein-cholesterol (HDL-C) (delta = - 0.07 mmol/ 1, p = 0.03) and a trend towards increased triglycerides (delta = 0.25 mmol/ 1, p = 0.07). In women, a trend towards the high triglyceride, low HDL-C phenotype was evident (delta = - 0.02 mmol/1 for HDL-C and delta = 0.14 mmol/l for triglycerides, respectively). Cumulative analysis of other studies of male carriers of the D9N and N291S revealed higher levels of triglycerides (D291N; 2.60(1.85) mmol/l vs. 1.62(1.18) mmol/l: p < 0.0001) (D9N; 1.94 (1.19) mmol/l vs. 1.74(1.17) mmol/l: p < 0.001) and lower HDL-C (N291S; 1.04(0.32) mmol/l vs. 1.15(0.28) mmol/l: p < 0.0001) (D9N; 1.08(0.24) mmol/l vs. 1.16(0.28) mmol/l: p < 0.0001). In females, results differed with higher TG levels (N291S; 1.70(0.99) mmol/l vs. 1.10(0.63) mmol/l: p < 0.001) (D9N; 1.08(0.76) mmol/l vs. 0.96(0.51) mmol/l: p < 0.01) and lower HDL-C levels (N291S; 1.27(0.33) mmol/l vs. 1.51(0.32) mmol/l: p < 0.0001); however, the HDL-C levels for D9N carriers were similar to non-carriers (D9N; 1.52(0.29) mmol/l vs. 1.53(0.35) mmol/l: p = 0.83). Our data provide evidence that common variants of the LPL gene are significant modulators of lipid and lipoprotein levels in both men and women.

Phenotypic variation in heterozygous familial hypercholesterolemia: a comparison of Chinese patients with the same or similar mutations in the LDL receptor gene in China or Canada.

Familial hypercholesterolemia (FH) is caused by mutations in the LDL receptor (LDLR) gene and is usually associated with hypercholesterolemia, lipid deposition in tissues, and premature coronary artery disease (CAD). However, individuals with heterozygous FH in China exhibit a milder phenotype despite having deleterious mutations in the LDLR gene (X.-M. Sun et al, Arterioscler Thromb. 1994;14:85-94). Nineteen Chinese FH heterozygotes living in Canada were screened for the 11 mutations that had been described in FH patients living in China. One Chinese Canadian carried one of these mutations (Trp462Stop), 2 carried a previously unreported single-base substitution (Cysl63Arg), and 1 carried a mutation observed in French-Canadian patients (Glu207Lys). Twelve additional carriers of these mutations were identified in the families of the index patients. Significantly higher LDL cholesterol concentrations were observed in FH heterozygotes with defined mutations living in Canada (mean+/-SD, 7.46+/-1.29, n=16) than in those living in China (4.35+/-1.09, n=18; P<.0001). Six of the 16 FH heterozygotes residingin Canada had evidence of tendon xanthomata and 4 had a history of premature CAD, whereas none of those in China had tendon xanthomata or CAD. Complete segregation between hypercholesterolemia and inheritance of a mutant allele was observed in 3 Canadian Chinese FH families. Thus, Chinese FH heterozygotes living in Canada exhibit a phenotype similar to that of other FH patients in Western societies. The difference between patients living in Canada and those living in China could be ascribed to differences in dietary fat consumption, showing that environmental factors such as diet play a significant role in modulating the phenotype of heterozygous FH.

A common mutation in the lipoprotein lipase gene (N291S) alters the lipoprotein phenotype and risk for cardiovascular disease in patients with familial hypercholesterolemia.

BACKGROUND: Recently, a mutation in the lipoprotein lipase (LPL) gene (N291S) has been reported in 2% to 5% of individuals in western populations and is associated with increased triglyceride (TG) and reduced HDL cholesterol (HDLC) concentrations. METHODS AND RESULTS: Here we report a significant alteration in biochemical and clinical phenotype in subjects with familial hypercholesterolemia (FH) who are heterozygous for this N291S LPL mutation. Sixty-four FH heterozygotes carrying the N291S mutation had significantly a higher TG level (P=.004), a higher ratio of total cholesterol to HDLC (P<.001), and lower HDLC concentrations (P=.002) compared with 175 FH heterozygotes without this LPL mutation. Moreover, the N291S mutation conferred a significantly greater risk for developing cardiovascular disease in FH heterozygotes compared with FH heterozygotes without this LPL mutation (odds ratio, 3.875; P=.006). CONCLUSIONS: These data provide evidence that a common LPL variant (N291S) significantly influences the biochemical phenotype and risk for cardiovascular disease in patients with FH.

Ethnic variation and in vivo effects of the -93t-->g promoter variant in the lipoprotein lipase gene.

Recently, a (t-->g) transition at nucleotide -93 in the lipoprotein lipase (LPL) gene promoter has been observed in Caucasians. Here, we have compared the frequency of the -93g carriers in three distinct populations (Caucasians, South African Blacks, and Chinese). The carrier frequency in the Caucasian population was 1.7% (4/232), which was in contrast to the South African Black population, which had a frequency for this allele of 76.4% (123/161) of the individuals tested. This transition was not observed in the Chinese population under study. Near complete linkage disequilibrium between the -93g and the previously described D9N mutation was observed in the Caucasian population but not in South African Blacks. To further assess the ancestral origins of these DNA changes, DNA haplotyping using a CA repeat 5' to these substitutions was performed. The -93t allele was associated with only a few specific dinucleotide repeat sizes. In contrast, the -93g allele occurred on chromosomes with many different repeat lengths. The broad distribution of repeats on -93g carrying chromosomes, their high frequency in the South African Black population, and the conservation of the -93g allele among different species may suggest that the -93g allele is the ancestral allele on which a transition to t and the D9N mutations arose. The very high frequency of the -93g allele distinct from the N9 allele in a cohort of Black South Africans allowed us to specifically assess the phenotypic effects of the -93g allele on lipids. Individuals homozygous for the g allele at -93 showed mildly decreased triglycerides compared with individuals homozygous for the t allele (1.14 +/- 0.66 mmol/L versus 0.82 +/- 0.3; P = .04). Thus, the -93g allele in this cohort is associated with low plasma triglyceride levels.

Differences in the phenotype between children with familial defective apolipoprotein B-100 and familial hypercholesterolemia.

Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited genetic disorder resulting from a point mutation in the apolipoprotein (apo) B gene and is associated with significantly elevated plasma total and LDL cholesterol levels. Despite numerous descriptions outlining the phenotype of children with familial hypercholesterolemia (FH), no study has described the biochemical and clinical phenotype in a cohort of children with FDB. The phenotypes of FH and FDB, therefore, have not been compared in children. We have studied a cohort of 38 Dutch children (all <20 years old) with FDB from 21 different families. Lipid and lipoprotein levels and the clinical phenotype were compared with 97 age-matched FH heterozygotes, as defined by molecular analysis, and with age-matched non-FDB, non-FH control subjects. Female FDB carriers (n=23) had significantly lower total cholesterol (P<.001), LDL cholesterol (P=.001), total cholesterol:HDL ratio (P<.001), and apoB levels (P=.001) than age-matched female FH heterozygotes (n=50). Similar results were noted in male FDB carriers (n=15) compared with male FH heterozygotes (n=47; P=.005, P=.007, P=.014, and P=.074, respectively). Within the FDB group, female FDB heterozygotes had higher LDL cholesterol (P=.038) and a trend to higher total cholesterol levels (P=.165) than age-matched males. Both male and female FDB carriers had significantly higher total cholesterol, LDL cholesterol, and total cholesterol:HDL ratio than age- and sex-matched control subjects, which was evident even in children <10 years of age, providing additional evidence that this mutation is penetrant in early life. These results provide evidence for a milder biochemical phenotype in children with FDB than in children with FH. The phenotype observed is intermediate between that of control subjects and FH heterozygotes matched for age and sex. As the incidence of coronary artery disease is related to both the extent and duration of cholesterol elevation, our findings might explain in part the lower incidence of clinical atherosclerosis seen in adults with this condition than in adults with FH.

Familial defective apolipoprotein B-100 in hypercholesterolemic Chinese Canadians: identification of a unique haplotype of the apolipoprotein B-100 allele.

Familial defective apo B-100 (FDB) is an autosomal dominant condition resulting in hypercholesterolemia. It is generally observed in 1-6% of hypercholesterolemic subjects in Caucasian populations studied. There are, thus far, no reports characterizing the frequency and phenotype of FDB in a Chinese population. We report on the frequency of the FDB (Arg(3500)--> Gln) mutation and the associated haplotype among 160 hypercholesterolemic (TC > or = 6.2 mmol/l) Chinese Canadians including 36 subjects with a clinical diagnosis of familial hypercholesterolemia (FH). Screening for the FDB mutation was done using a mutagenic polymerase chain reaction and haplotype analysis was undertaken using eight diallelic markers and the 3'HVR marker. One Chinese Canadian clinical FH heterozygote was positive for the FDB Arg(3500)--> Gln mutation while none of the remaining non-FH hypercholesterolemic subjects (n = 124) were carriers of this mutation. Haplotype analysis in the patient positive for this mutation revealed a unique haplotype which differed from both the common haplotype of this mutation observed in Caucasians and from the only other haplotype reported in a Chinese individual. The associated haplotype included a 9-base pair deletion in the signal peptide region and the presence of three restriction sites absent in previously reported haplotypes. These data suggest that the apo B-100 Arg(3500)--> Gln mutation does not appear to be a significant factor contributing to moderate hypercholesterolemia in a Chinese population residing in Canada. However, this mutation was rarely observed among Chinese individuals with a clinical diagnosis of FH. The presence among Chinese individuals of two different haplotypes associated with this mutation, which are different from what has been described among Caucasians is compatible with multiple recurrent origins for this mutation in the Chinese population.

A frequently occurring mutation in the lipoprotein lipase gene (Asn291Ser) results in altered postprandial chylomicron triglyceride and retinyl palmitate response in normolipidemic carriers.

An Asn291Ser mutation in exon 6 of the lipoprotein lipase gene (LPL) frequently occurs in Caucasians (2-4%) and results in a partial catalytic defect. Although this mutation may be associated with low HDL cholesterol and elevated triglyceride levels, some carriers are normolipidemic and may have LPL activity in the normal range in the fasting state. To assess in vivo the influence of dietary stress on the function of this mutation, we have performed oral fat load studies on three unrelated normolipidemic Asn291Ser carriers and compared these results to five healthy controls and to a subject with a clear 50% reduction in LPL activity compared with controls. The Asn291Ser carriers exhibited a more pronounced postprandial response compared with non-carriers as evidenced by higher chylomicron triglyceride (TG) and chylomicron retinyl palmitate peaks (P = 0.03 and P = 0.02, respectively). Significantly higher area under response curves were also seen for both chylomicron triglycerides (P = 0.02) and chylomicron retinyl palmitate (P = 0.01) when compared with non-carriers. These results provide further in vivo evidence for the functional effects of this common mutation despite normal fasting lipid levels. These data suggest that even though subjects with this mutation may be normolipidemic in the fasting state, environmental stress such as an oral fat load may unmask the catalytic defect and result in significant disturbances in postprandial chylomicron metabolism.

Mutations in the gene for lipoprotein lipase. A cause for low HDL cholesterol levels in individuals heterozygous for familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is characterized by elevated plasma concentrations of LDL cholesterol resulting from mutations in the gene for the LDL receptor. Low HDL cholesterol levels are seen frequently in patients both heterozygous and homozygous for mutations in this gene. Suggested mechanisms for reduced HDL levels in FH patients have been altered apolipoprotein A-1 metabolism and elevated cholesteryl ester transfer protein activity, but the molecular basis for hypoalphalipoproteinemia in any of these patients has not yet been identified. We investigated four large families in which individuals were found to be double heterozygotes for both FH and lipoprotein lipase (LPL) deficiency. These double heterozygotes have significantly less HDL cholesterol than persons with FH or LPL heterozygosity alone. In the double heterozygotes, HDL particle composition is not significantly different from FH heterozygotes, suggesting a quantitative rather than qualitative defect in HDL metabolism in these persons. We propose that mutations in the LPL gene may be a cause of low HDL cholesterol levels in some individuals heterozygous for FH.