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Fibrodysplasia ossificans progressiva (FOP) is a rare but very severe disease, characterised by congenital malformations of the toes and by progressive heterotopic ossification of muscles and joints. Two genes, the noggin (NOG) gene and the activin A type I receptor (ACVRI) gene, are involved in FOP. In this study we have searched for the NOG and the 617G>A (ACVR1) mutations in a well characterized series of twenty-seven French FOP patients. Five NOG mutations (delta 42, 274G>C, 275G>A, 276G>A, and 283G>A) have been found in seven (26%) of our FOP patients. The 617G>A mutation in the ACVR1 gene is found in fourteen (52%) of the patients. With one exception (patient number 22), 617G>A and NOG mutations are mutually exclusive in patients. Mutations 274G>C, 283G>A and 617G>A segregate with the trait in five different FOP families, some members of them being partially affected by the disease.

Familial essential tremor (ET), the most common inherited movement disorder, is generally transmitted as an autosomal dominant trait. A genome-wide scan for ET revealed one major locus on chromosome 3q13. Here, we report that the Ser9Gly variant in the dopamine D(3) receptor gene (DRD3), localized on 3q13.3, is associated and cosegregates with familial ET in 23 out of 30 French families. Sequencing revealed no other nonsynonymous variants in the DRD3-coding sequence and in the first 871 bp of the 5' flanking region. Moreover, Gly-9 homozygous patients presented with more severe and/or earlier onset forms of the disease than heterozygotes. A replication study comparing 276 patients with ET and 184 normal controls confirmed the association of the Gly-9 variant with risk and age-at-onset of ET. In human embryonic kidney (HEK) 293-transfected cells, the Gly-9 variant did not differ from the Ser-9 variant with respect to glycosylation and to anterograde and retrograde trafficking, but dopamine had an affinity that was four to five times higher. With the Gly-9 variant, the dopamine-mediated cAMP response was increased, and the mitogen-associated protein kinase (MAPK) signal was prolonged, as compared with the Ser-9 variant. The gain-of-function produced by the Gly-9 variant may explain why drugs active against tremor in Parkinson's disease (PD) are usually not effective in the treatment of ET and suggests that DRD3 partial agonists or antagonists should be considered as novel therapeutic options for patients with ET.

Cholesteryl ester transfer protein (CETP) plays a key role in the determination of high-density lipoprotein (HDL) levels via its action on intravascular HDL metabolism. The TaqIB polymorphism of the CETP gene is associated with plasma CETP and high-density lipoprotein cholesterol (HDL-C) levels and with premature coronary artery disease. Such associations appear to result from linkage disequilibrium between TaqIB and other functional polymorphisms. To date, only one functional promoter variant, which may explain the effects of TaqIB, has been identified at position -629 in the CETP gene. Here we describe a C/T polymorphism located at position -1337 in the human CETP gene (C allele frequency: 0.684), which is significantly associated with plasma HDL-C and CETP levels (P=0.0001 and P<0.0001, respectively). Transient transfection of a reporter gene construct containing the CETP promoter from -1707/+28 in liver cells (HepG2) revealed that the -1337T allele was expressed to a significantly lower degree (-34%, P<0.0001) than the -1337C allele. In addition, we clearly demonstrated that the -971G/A polymorphism is functional and that its functionality is intimately linked to the presence of the -1337 site. In vitro evaluation of potential interaction between -1337C/T and other functional variants of the CETP gene (-971G/A and -629C/A) demonstrated that these three functional CETP promoter polymorphisms can interact together to determine the overall activity of the CETP gene and thus contribute significantly to variation in plasma CETP mass concentration.

Objective: To screen for mutations in the GnRH receptor gene in a case of complete hypogonadotropic hypogonadism (HH) with GnRH resistance., Design: Case report., Setting: A university hospital., Patient(s): A male patient with the complete form of HH without anosmia., Intervention(s): Physical examination and laboratory and genetic studies., Main Outcome Measure(s): Gonadotropins at the basal state and after GnRH administration and GnRH receptor DNA sequencing., Result(s): A novel missense mutation, localized in the first amino acid of the extracellular loop found in the heterozygous state, and another mutation, Arg(139)His (R139H), located in the conserved aspartate-arginine-serine motif at the junction of the third transmembrane and second intracellular loop of the GnRH receptor, were identified in the homozygous state. Pedigree studies reveal that both parents were heterozygous for R139H, while the mother carried the missense mutation at codon 1(M1T)., Conclusion(s): GnRH receptor mutations may account for a larger proportion of cases of HH than previously thought. The phenotypic spectrum of HH seems to vary, and this heterogeneity may be related, at least in part, to the degree of impaired biological activity of the mutated GnRH receptor caused by the allelic type of mutations.

Objective: To report the occurrence of a spontaneous pregnancy in a patient who was homozygous for the Q106R mutation in the GnRH receptor (GnRHR) gene., Design: Case report., Setting: Reproductive endocrinology unit of an academic medical center., Patient(s): A 27-year-old woman who initially presented with partial idiopathic hypogonadotropic hypogonadism and who achieved a spontaneous pregnancy 3 months after oral contraceptive pill (OCP) withdrawal., Intervention(s): Blood sampling for hormonal and genetic investigations, transvaginal ultrasound., Main Outcome Measure(s): LH, FSH, E2, and betahCG serum levels. Ultrasound examination of the uterine cavity., Result(s): Three months after OCP withdrawal, the patient was amenorrheic. However, the betahCG serum level was 149 IU/L. Transvaginal ultrasound 2 weeks later revealed the presence of one intrauterine sac containing two embryos with cardiac activity. At 9 weeks of gestation, no cardiac activity was found. A curettage was then performed, and the pathological examination indicated the presence of chorionic villi., Conclusion(s): OCP withdrawal might have induced a transient situation with optimal endogenous pulsatile GnRH secretion, thus overriding the GnRH resistance induced by the partially inactivating Q106R GnRHR gene mutation and allowing ovulation to occur.

GCV-ablation of transplanted TK-positive liver tumors or the application of syngenic and allogenic HSV-TK/GCV oncolysates significantly reduced the size of synchronously growing untreated sister tumors in the liver. These TK-negative liver tumors constantly showed an increased infiltration by mononuclears (x4). The relative abundance of CD 4/8, NK and monocyte subtypes remained constant. The distant bystander effect was associated with a strong induction of GMCSF and IL-12 expression in the untreated TK-negative liver tumors. Analysis of the vbeta T-cell receptor profiles from TK-negative tumors did not point to clonal lymphocyte expansions. These results support the view of the 'distant bystander effect' as a predominantly local phenomenon, which is mediated by resident immune effectors rather than by MHC I restricted CD 3 positive lymphocytes.

Activating mutations of the calcium-sensing receptor (CaR) can cause isolated hypoparathyroidism. Treatment of hypocalcemia in these patients remains to be optimized, because the use of 1-hydroxylated vitamin D3 derivatives can cause hypercalciuria and nephrocalcinosis. We identified activating CaR mutations in 8 (42%) of 19 unrelated probands with isolated hypoparathyroidism. The severity of hypocalcemic symptoms at diagnosis was independent of age, mutation type, or mode of inheritance but was related to the degree of hypocalcemia; serum Ca was 1.97 +/- 0.08, 1.82 +/- 0.14, and 1.54 +/- 0.22 mmol/liter, respectively, in asymptomatic (n = 7), mildly symptomatic (n = 8), and severely symptomatic patients (n = 6). Hypocalcemia segregated with the CaR mutation, but no phenotype-genotype relationships were identified. Fourteen patients received regular 1-hydroxylated vitamin D3 treatment (mean duration, 7.2 +/- 4.9 yr). Nine had hypercalciuric episodes, which were associated with nephrocalcinosis in eight cases. Serum Ca during treatment predicted hypercalciuria and nephrocalcinosis poorly, because either or both of the latter could develop in hypocalcemic patients. Thus, mutational analysis of the CaR gene should be considered early in the work-up of isolated hypoparathyroidism. Treatment options should be weighed carefully in patients with serum Ca below 1.95 mmol/liter. The risk of nephrocalcinosis during treatment can be minimized by carefully monitoring urinary Ca excretion.

After serial passage in the presence of increasing concentrations of ganciclovir (GCV) in vitro, a human herpesvirus-6 (HHV-6) mutant exhibiting a decreased sensitivity to the drug was isolated. Analysis of drug susceptibility showed that the IC(50) of this mutant was 24-, 52- and 3-fold higher than that of the wild-type (wt) IC(50) in the case of GCV, cidofovir and foscarnet, respectively. Genotypic analysis showed two single nucleotide changes as compared to the wild-type: an A-->G substitution of the U69 protein kinase (PK) gene resulted in an M(318)V amino acid substitution and the other change, located in the C-terminal part of the U38 gene, resulted in an A(961)V amino acid substitution within the DNA polymerase. The M(318)V change was located within the consensus sequence DISPMN of the putative catalytic domain VI of the PK. This change was homologous to the M(460)V and M(460)I changes that had been reported previously within the consensus sequence DITPMN of the human cytomegalovirus (HCMV) UL97 PK and associated with the resistance of HCMV to GCV. The M(318)V change was also detected by PCR in HHV-6-infected PBMCs from an AIDS patient who had been treated with GCV for a long period of time and exhibited a clinically GCV-resistant HCMV infection. These findings provide strong circumstantial evidence that the M(318)V change of the PK gene is associated with resistance to GCV and raise the question of cross resistance to this drug among different betaherpesviruses.

We describe a woman with complete hypogonadotropic hypogonadism and a new compound heterozygous mutation of the GnRH receptor (GnRHR) gene. A null mutation L314X leading to a partial deletion of the seventh transmembrane domain of the GnRHR is associated with a Q106R mutation previously described. L314X mutant receptor shows neither measurable binding nor inositol phosphate production when transfected in CHO-K1 cells compared to the wild-type receptor. The disease is transmitted as an autosomal recessive trait, as shown by pedigree analysis. Heterozygous patients with GnRHR mutations had normal pubertal development and fertility. The present study shows an absence of LH and FSH response to pulsatile GnRH administration (20 microg/pulse, sc, every 90 min). However, GnRH triggered free alpha-subunit (FAS) pulses of small amplitude, demonstrating partial resistance to pharmacological doses of GnRH. FSH, LH, and FAS concentrations were evaluated under chronic estrogen treatment and repeat administration of GnRH. Not only were plasma FSH, LH, and FAS concentrations decreased, but FAS responsiveness was reduced. This new case emphasizes the implication of the GnRH receptor mutations in the etiology of idiopathic hypogonadotropic hypogonadism. We also have evidence for a direct negative estrogen effect on gonadotropin secretion at the pituitary level, dependent on the GnRHR signaling pathway.

Autosomal dominant hypocalcemia (ADH) can result from heterozygous missense activating mutations of the calcium-sensing receptor (CaSR) gene, a G-protein-coupled receptor playing key roles in mineral ion metabolism. We now describe an ADH kindred of three generations caused by a novel CaSR mutation, a large in-frame deletion of 181 amino acids within its carboxylterminal-tail from S895 to V1075. Interestingly, the affected grandfather is homozygous for the deletion but no more severely affected than heterozygous affected individuals. Functional properties of mutant and wild-type (WT) CaSRs were studied in transiently transfected, fura-2-loaded human embryonic kidney (HEK293) cells. The mutant receptor exhibited a gain-of-function, but there was no difference between cells transfected with mutant complementary DNA alone or cotransfected with mutant and WT complementary DNAs, consistent with the similar phenotypes of heterozygous and homozygous family members. Therefore, this activating deletion may exert a dominant positive effect on the WT CaSR. The mutant receptor's cell surface expression was greater than that of the WT CaSR, potentially contributing to its gain-of-function. This novel mutation in the CaSR gene provides the first known examples of a large naturally occurring deletion within a G-protein-coupled receptor's carboxylterminal-tail and of a homozygous, affected individual with ADH.

The expression pattern of the GnRH receptor was investigated in a variety of normal and neoplastic human tissues by RT-PCR-Southern blotting. In addition to the full-length cDNA (sb1), we identified two other transcripts: the first (sb2) was characterized by a 128 bp deletion as previously described; the second was an unexpected finding composed of a shorter cDNA (sb3), the sequence of which revealed a 220 bp deletion corresponding in size to exon 2. These three transcripts were found in normal pituitary and pituitary adenomas, and in granulosa tumors, but not in testis, where sb2 was lacking. Only sb1 was expressed in normal, fibrocystic and malignant breast tissue. No transcript with a full-length region was found in endometrium, intestine or lymphocytes. This is the first report that shows that splicing of the gonadotropin-releasing hormone receptor gene is tissue dependent. We also determined the intron-exon nucleotide sequence of the gene and identified an MaeIII polymorphic site in exon 1 created by a silent C453T transition found in 10% of unrelated French whites.

Our purpose was to search for mutations in the apolipoprotein E (apoE) gene and to evaluate the role of apoE polymorphisms in the occurrence of autosomal dominant retinitis pigmentosa (ADRP). The ApoE gene coding sequence was analyzed in 51 unrelated patients affected with ADRP. A screening for mutations by SSCP and an analysis of the apoE polymorphisms were performed using PCR and restriction enzymatic digestion. No abnormal patterns of migration were observed by SSCP analysis. No significant statistical difference was seen between our ADRP population and the French general population for apoE allele frequency. From these results we report that the apoE gene does not seems to be involved in our ADRP population.

Objective: Given the central role of the GnRH receptor (GnRHR) in the regulation of the gonadotrophin secretion, it might be implicated directly or indirectly in the pathogenesis of gonadotroph tumours., Design: We determined if GnRHR mRNA was expressed in gonadotroph tumours using RT-PCR and analysed the GnRHR gene for the presence of mutations in its coding region, using direct sequencing of PCR products. Results were analysed according to the pattern of expression of alpha, beta-FSH and beta-LH subunit (SU) genes., Subjects: RNA was extracted from 20 gonadotroph tumours identified by immunohistochemistry (> 10% of stained cells): 9 adenomas were functioning (high serum gonadotrophin levels), 3 were associated with high alpha-SU levels and 8 were nonfunctioning. Genomic DNA was extracted from 64 normal subjects., Results: We found GnRHR mRNA in 12 tumours (60%): 8/9 functioning (88%), 1/3 alpha-secreting (33%) and 3/8 nonfunctioning (37.5%) gonadotroph adenomas. There was a significant association between GnRHR expression and immunostaining for beta-FSH (P = 0.014). The nucleotide sequence of the amplified products was identical to that of human pituitary except for the presence, in 3 functioning adenomas, of a silent C to T transition at nucleotide 453 encoding for the serine residue situated in the second intracellular loop at position 151. Heterozygosity provided evidence that both alleles were transcribed in these tumours. This substitution creates a Mae III restriction site. Genomic DNA from normal subjects were then tested for the presence of this new polymorphism. The frequency of the heterozygosity (18.7%) was not significantly different from that found in gonadotroph tumours (25%) and this new Mae III polymorphism site cannot be used as a tumoural marker., Conclusion: The GnRHR gene is preferentially expressed in functioning rather than in nonfunctioning gonadotroph adenomas, but no mutations altering the coding region of the gene were found to further substantiate its role in the pathogenesis of gonadotroph tumours.

Purpose: Apolipoprotein E (ApoE) is a polymorphic protein that plays a central part in plasma metabolism of lipids and in central nervous system lipid homeostasis. Our purpose was to evaluate the potential role of ApoE polymorphism in the occurrence of exudative age-related macular degeneration associated with drusen, which contain lipids., Methods: We analyzed apolipoprotein E genotypes in 116 unrelated patients with exudative age-related macular degeneration in one eye and hard drusen (n = 39) or soft drusen (n = 77) in the other eye, and compared the results with those of age-matched and sex-matched control subjects (n = 168). Apolipoprotein E alleles were detected by a ploymerase chain reaction-based method., Results: A lower frequency of the epsilon4 allele carriers was observed in the exudative age-related macular degeneration group compared with control subjects (12.1% vs 28.6%, respectively; P < .0009). The epsilon4 allele was less frequent in the age-related macular degeneration group compared with control subjects (0.073 vs 0.149, respectively; P < .006). This decreased frequency of the epsilon4 allele was mainly observed in the soft drusen subgroup compared with control subjects (0.045 vs 0.149, respectively; P < .0009)., Conclusion: This lower relative frequency of the epsilon4 allele supports the hypothesis that the ApoE gene is a genetic protective factor identified in age-related macular degeneration.

Using DNA sequencing of the coding and exon flanking regions of the low density lipoprotein receptor (LDLR) gene we identified an Alw26 I site in exon 10 by a transition G142A. The alleles are represented by one uncut fragment (A1=108bp) or two fragments (A2=82bp and 26 pb). Two other fragments (72bp and 16bp) were systematically found within the amplified product. The alleles were detected in 157 unrelated French Caucasians with A1 frequency = 0.58 and A2=0.42. The observed heterozygoty was 44.5%. Homozygous familial hypercholesterolemie (FH) has a severe clinical picture leading to death during childhood. Because it is very informative, the present polymorphism was very useful as genetic marker for clinical diagnois and counseling as we described in linkage analysis at the LDLR locus for prenatal diagnosis in a fetus who could inherit two LDLR mutant alleles from FH heterozygote parents.

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.

Mutations in the LPL gene show high levels of allelic heterogeneity between and within different populations. Complete LPL deficiency has a very high prevalence in French Canadians, where only three missense mutations account for > 97% of cases, most consistent with founder mutations introduced early in Quebec by French immigrants. In order to determine whether these mutations were present in France, 12 unrelated French families with defined LPL deficiency were investigated for the presence of the mutations found in French Canadians. Of the 24 expected alleles, six (25%) represented mutations in French Canadians (Gly188Glu four alleles, Asp250Asn and Pro207Leu one allele each). Comparison of French Canadian and French alleles identified the same haplotype in all carriers of the Gly188Glu and of the Asp250Asn, suggesting a common origin. In contrast, the Pro207Leu occurred on different haplotypes in France and Quebec, compatible with a different ancestral origin.

Apolipoprotein E genotypes (alleles *2, *3, and *4) have been determined in 70 Aka Pygmies and 470 unrelated African sub-Saharan subjects. Allele frequencies for Pygmies are 5.7% for APOE*2, 53.6% for APOE*3, and 40.7% for APOE*4, and the global proportions for sub-Saharan subjects are 11.6% for APOE*2, 70.6% for APOE*3, and 17.8% for APOE*4. The frequencies in some ethnic groups are statistically different from the overall mean in the Afar and the Isa, the Ewe (Togo), the Malinke (Guinea), and the Mossi; three ethnic groups have a higher allele frequency of APOE*4 (Fon, 29.4%; Zairians, 33.3%; Tutsi, 38.5%). The APOE*4 allele is considered the ancestral form because of its high frequency in African Pygmies and other aboriginal populations.

While gonadotropin-releasing hormone (GnRH) or GnRH receptor (GnRHR) have been reported to exist in tissues other than brain and pituitary, there is no report concerning co-expression of GnRH and GnRHR in human breast tissues. To address this question, we have examined whether mRNA for GnRH as well as GnRHR was present in different human breast samples, by employing the reverse transcription-polymerase chain reaction (RT-PCR) protocol followed by Southern blotting of the PCR products. Coexpression of GnRH and GnRHR genes was further confirmed by dot blot hybridization using appropriate [32P]-labeled probes. We thus tested fibrocystic breast (4 cases), invasive ductal carcinomas (6 cases) and 1 adjacent non-neoplastic tissue. GnRHR and GnRH mRNAs were found in all actin-positive samples including malignant as well as nonmalignant tissues. Quantitative determinations of mRNA did not reveal significant differences between malignant and non-malignant breast samples for either GnRH or GnRHR gene expression. Our data show that neither gene was overexpressed in the breast cancer samples compared with normal breast tissue. Since GnRH agonists inhibit breast epithelial cell growth, the presence of GnRHR mRNA suggests that GnRH may specifically affect breast cell growth. Our data thus raise the possibility of an autocrine/paracrine role for GnRH in human mammary gland.

Variability in the expression of monogenic lipid disorders may be observed in patients carrying the same DNA mutation, suggesting possible genetic or environmental interactions. Our objective was to investigate the genotype-phenotype relationships in two unrelated French patients with an aggravated expression of a dominantly inherited hypercholesterolemia. In probands, segregation analysis complemented by DNA sequencing identified heterozygous defective alleles and mutations on two nonallelic loci for two monogenic lipid disorders: familial hypercholesterolemia at the low density lipoprotein (LDL) receptor locus and familial defective apolipoprotein B-100 at the locus encoding its ligand, apolipoprotein B-100. The LDL-receptor missense mutations had been reported in French Canadians. The apolipoprotein B mutation was the Arg3500Gln founder mutation in Northern Europe. Probands had an unusual phenotype of aggravated hypercholesterolemia that was complicated with premature coronary arterial disease, although remaining responsive to lipid-lowering drugs. This phenotype was distinct from that observed in their heterozygous relatives and distinct from those observed in FH or FDB homozygotes. These cases refer to a new class of patients with digenic lipid disorders, defined by specific clinical features that result from the combined effects of two independent loci. Moreover, the observed phenotype of aggravated hypercholesterolemia gives further evidence that receptor and ligand play distinct roles in regulating LDL metabolism. Although uncommon, these cases give insight into the molecular mechanisms that underly the clinical variability of inherited hypercholesterolemia.