Ursodeoxycholic acid in intrahepatic cholestasis of pregnancy: a secondary analysis of the PITCHES trial.

OBJECTIVE: To evaluate whether a particular group of women with intrahepatic cholestasis of pregnancy (ICP), based on their presenting characteristics, would benefit from treatment with ursodeoxycholic acid (UDCA). DESIGN: Secondary analysis of the PITCHES trial (ISRCTN91918806). SETTING: United Kingdom. POPULATION OR SAMPLE: 527 women with ICP. METHODS: Subgroup analyses were performed to determine whether baseline bile acid concentrations or baseline itch scores moderated a woman's response to treatment with UDCA. MAIN OUTCOME MEASURES: Bile acid concentration and itch score. RESULTS: In women with baseline bile acid concentrations less than 40 µmol/l, treatment with UDCA resulted in increased post-randomisation bile acid concentrations (geometric mean ratio 1.19, 95% CI 1.00-1.41, P = 0.048). A test of interaction showed no significance (P = 0.647). A small, clinically insignificant difference was seen in itch response in women with a high baseline itch score (-6.0 mm, 95% CI -11.80 to -0.21, P = 0.042), with a test of interaction not showing significance (P = 0.640). Further subgroup analyses showed no significance. Across all women there was a weak relationship between bile acid concentrations and itch severity. CONCLUSIONS: There was no subgroup of women with ICP in whom a beneficial effect of treatment with UDCA on bile acid concentration or itch score could be identified. This confirms that its routine use in women with this condition for improvement of bile acid concentration or itch score should be reconsidered. TWEETABLE ABSTRACT: PITCHES: No group of women with ICP has been found in whom UDCA reduces bile acid concentrations or pruritus.

Cholestatic pregnancy is associated with reduced placental 11ßHSD2 expression.

INTRODUCTION: Intrahepatic Cholestasis of Pregnancy (ICP) is associated with an increased risk of fetal morbidity and mortality and is characterised by elevated bile acids in the maternal and fetal compartments. Bile acids have been shown to attenuate renal 11ßHSD2 expression and, given the protective role of placental 11ßHSD2 in preventing fetal exposure to excessive maternal cortisol, we aimed to establish whether raised serum bile acids in ICP influence placental 11ßHSD2 expression. METHODS: Placental tissue from human and murine cholestatic pregnancy was evaluated for changes in 11ßHSD2 mRNA expression compared to uncomplicated pregnancy using quantitative PCR. Parallel in vitro studies were performed using BeWo choriocarcinoma cells to assess the effect of different bile acid species on 11ßHSD2 gene expression and whether concurrent UDCA administration can reverse any bile acid induced changes. RESULTS: Placental 11ßHSD2 mRNA expression was reduced in human and murine cholestatic pregnancy. In BeWo cells, treatment with the primary bile acid CDCA resulted in reduced 11ßHSD2 gene expression, while treatment with other primary bile acids had no significant effect. Furthermore, the tertiary bile acid UDCA, used in the treatment of ICP did not significantly affect 11ßHSD2 mRNA levels either alone, or when co-administered with CDCA. DISCUSSION: Under cholestatic conditions placental 11ßHSD2 mRNA is reduced. Studies in BeWo choriocarcinoma cells demonstrated that CDCA is likely to be the specific bile acid that mediates this effect. UDCA, the main bile acid used to treat cholestasis, did not reduce placental 11ßHSD2 expression, further supporting its use in the management of ICP.

A placental phenotype for intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy specific liver disease associated with significant risk of fetal complications. It is hypothesised that the risk of adverse fetal outcomes relates to the toxic effects of bile acids, the levels of which are increased in both maternal and fetal serum. Human and rodent studies have shown that transplacental transfer of bile acids is impaired in ICP. Furthermore, the morphology of placentas from the rodent model of ICP is markedly abnormal, and is associated with increased expression of apoptotic markers and oxidative stress. Using placental tissue from ICP cases and normal pregnancies and cultured placental explant fragments we investigated the histological and molecular effects of cholestasis. We also examined the influence of ursodeoxycholic acid (UDCA) administration on these parameters. Here we report that ICP is associated with several morphological abnormalities of the placenta, including an increase in the number of syncytial knots, and that these can be reproduced in an in vitro (explant) model exposed to the bile acids taurocholic acid and taurochenodoexycholic acid. Furthermore, we demonstrate that ursodeoxycholic acid, a drug commonly used in the management of ICP, has a protective effect on placental tissue both in vivo and in vitro.

Characterisation of the nuclear receptors FXR, PXR and CAR in normal and cholestatic placenta.

Bile acids are the toxic end products of hepatic cholesterol metabolism. They are synthesised from early in gestation and excreted via the placenta. The mechanism for transplacental excretion of bile acids is not known. The gene and protein expression of the nuclear receptors responsible for hepatic bile acid metabolism and transport was studied in eight normal and fourteen cholestatic placentas, and in an ex vivo model. The expression of the nuclear receptor FXR and several of it's target genes and of PXR and CAR was found to be very low in both normal and cholestatic placenta.

Identification of 13 novel NLRP7 mutations in 20 families with recurrent hydatidiform mole; missense mutations cluster in the leucine-rich region.

BACKGROUND: NLRP7 (NALP7) has recently been identified as the causative gene for familial recurrent hydatidiform mole (FRHM), a rare autosomal recessive condition in which affected women have recurrent molar pregnancies of diploid biparental origin. To date only a small number of affected families have been described. Our objectives were to investigate the diversity of mutations and their localisation to one or both isoforms of NLRP7, by screening a large series of women with FRHM and to examine the normal expression of NLRP7 in ovarian tissue. METHODS: Fluorescent microsatellite genotyping of molar tissue was used to establish a diagnosis of FRHM. Twenty families were subsequently screened for mutations in NLRP7 using DNA sequencing. Expression of NLRP7 in the ovary was examined by immunohistochemical staining. RESULTS: 16 different mutations were identified in the study, 13 of which were novel. Missense mutations were found to be present in transcript variant 2 of NLRP7 and cluster in the leucine-rich region (LRR). A man with two affected sisters and homozygous for the p.R693P mutation had normal reproductive outcomes. In the normal human ovary, NLRP7 expression is confined to the oocytes and present at all stages from primordial to tertiary follicles. CONCLUSION: 13 novel mutations in NLRP7 were identified. We confirm that mutations in NLRP7 affect female but not male reproduction, and provide evidence that transcript variant 2 of NLRP7 is the important isoform in this condition. The mutation clustering seen confirms that the LRR is critical for normal functioning of NLRP7.

Contribution of variant alleles of ABCB11 to susceptibility to intrahepatic cholestasis of pregnancy.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) has a complex aetiology with a significant genetic component. ABCB11 encodes the bile salt export pump (BSEP); mutations cause a spectrum of cholestatic disease, and are implicated in the aetiology of ICP. METHODS: ABCB11 variation in ICP was investigated by screening for five mutant alleles (E297G, D482G, N591S, D676Y and G855R) and the V444A polymorphism (c.1331T>C, rs2287622) in two ICP cohorts (n = 333 UK, n = 158 continental Europe), and controls (n = 261) for V444A. PCR primers were used to amplify and sequence patient and control DNA. The molecular basis for the observed phenotypes was investigated in silico by analysing the equivalent residues in the structure of the homologous bacterial transporter Sav1866. RESULTS: E297G was observed four times and D482G once. N591S was present in two patients; D676Y and G855R were not observed. The V444A polymorphism was associated with ICP (allelic analysis for C vs T: OR 1.7 (95% CI 1.4 to 2.1, p<0.001)). In addition, CC homozygotes were more likely to have ICP than TT homozygotes: OR 2.8 (95% CI 1.7 to 4.4 p<0.0001). Structural analyses suggest that E297G and D482G destabilize the protein fold of BSEP. The molecular basis of V444A and N591S was not apparent from the Sav1866 structure. CONCLUSIONS: Heterozygosity for the common ABCB11 mutations accounts for 1% of European ICP cases; these two mutants probably reduce the folding efficiency of BSEP. N591S is a recurrent mutation; however, the mechanism may be independent of protein stability or function. The V444A polymorphism is a significant risk factor for ICP in this population.

Sequencing and functional assessment of hPXR (NR1I2) variants in intrahepatic cholestasis of pregnancy.

1. The purpose of this study was to evaluate the role of coding variation in hPXR (NR1I2) in intrahepatic cholestasis of pregnancy (ICP) and to functionally asses the response of PXR variants to ligands of interest in ICP. 2. The coding region of hPXR was sequenced in a cohort of 121 Caucasian ICP patients and exon 2 was sequenced in an additional 226 cases. Reporter assays were used to evaluate the function of all known hPXR variants in response to the secondary bile acid lithocholic acid and therapeutic agents rifampicin, ursodeoxycholic acid and dexamethasone. 3. Two coding single nucleotide polymorphisms (C79T and G106A) were detected in the ICP cohort at frequencies consistent with healthy populations. These do not alter hPXR function in response to ligands of interest to ICP. Analysis of all known coding hPXR variants demonstrates that while subtle changes in experimental design mask or may unveil the functional effects of genetic variation, these are not maintained in a standard functional assay. 4. Coding genetic variation in hPXR does not contribute to the aetiology of ICP in Caucasian populations.

The molecular genetics of intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP), also known as obstetric cholestasis, causes maternal pruritus and liver impairment, and may be complicated by spontaneous preterm labour, fetal asphyxial events and intrauterine death. Our understanding of the aetiology of this disease has expanded significantly in the last decade due to a better understanding of the role played by genetic factors. In particular, advances in our knowledge of bile homeostasis has led to the identification of genes that play a considerable role in susceptibility to ICP. In this review we consider these advances and discuss the disease in the context of bile synthesis and metabolism, focusing on the genetic discoveries that have shed light on the molecular aetiology and pathophysiology of the condition.

Mutations in the X-linked filamin 1 gene cause periventricular nodular heterotopia in males as well as in females.

Periventricular heterotopia (PH) is a human neuronal migration disorder in which many neurons destined for the cerebral cortex fail to migrate. Previous analysis showed heterozygous mutations in the X-linked gene filamin 1 (FLN1), but examined only the first six (of 48) coding exons of the gene and hence did not assess the incidence and functional consequences of FLN1 mutations. Here we perform single-strand conformation polymorphism (SSCP) analysis of FLN1 throughout its entire coding region in six PH pedigrees, 31 sporadic female PH patients and 24 sporadic male PH patients. We detected FLN1 mutations by SSCP in 83% of PH pedigrees and 19% of sporadic females with PH. Moreover, no PH females (0/7 tested) with atypical radiographic features showed FLN1 mutations, suggesting that other genes may cause atypical PH. Surprisingly, 2/24 males analyzed with PH (9%) also carried FLN1 mutations. Whereas FLN1 mutations in PH pedigrees caused severe predicted loss of FLN1 protein function, both male FLN1 mutations were consistent with partial loss of function of the protein. Moreover, sporadic female FLN1 mutations associated with PH appear to cause either severe or partial loss of function. Neither male could be shown to be mosaic for the FLN1 mutation in peripheral blood lymphocytes, suggesting that some neurons in the intact cortex of PH males may be mutant for FLN1 but migrate adequately. These results demonstrate the sensitivity and specificity of DNA testing for FLN1 mutations and have important functional implications for models of FLN1 protein function in neuronal migration.

Identification of a novel primary torsion dystonia locus (DYT13) on chromosome 1p36 in an Italian family with cranial-cervical or upper limb onset.

Primary torsion dystonia (PTD) is a clinically and genetically heterogeneous group of movement disorders, usually inherited in an autosomal dominant fashion. Three PTD loci (DYT1, DYT6 and DYT7) have been identified to date. However, in several PTD families linkage to the known loci has been excluded. We identified an Italian PTD family with 11 definitely affected members. Phenotype was characterised by juvenile or early-adult onset, prominent cranial-cervical and upper limb involvement, mild course and occasional generalisation. A genome-wide search performed in the family identified a novel PTD locus (DYT13) within a 22-cM interval on the short arm of chromosome 1, with a maximum lod score of 3.44 (theta = 0) between the disease and marker D1S2667.

DYT13, a novel primary torsion dystonia locus, maps to chromosome 1p36.13--36.32 in an Italian family with cranial-cervical or upper limb onset.

Primary torsion dystonia (PTD) is a clinically and genetically heterogeneous group of movement disorders, usually inherited in an autosomal dominant fashion with reduced penetrance. The DYT1 gene on chromosome 9q34 is responsible for most cases of early limb-onset PTD. Two other PTD loci have been mapped to date. The DYT6 locus on chromosome 8 is associated with a mixed phenotype, whereas the DYT7 locus on chromosome 18p is associated with adult onset focal cervical dystonia Several families have been described in which linkage to the known PTD loci have been excluded. We identified a large Italian PTD family with 11 definitely affected members. Phenotype was characterized by prominent cranial-cervical and upper limb involvement and mild severity. A genome-wide search was performed in the family. Linkage analysis and haplotype construction allowed us to identify a novel PTD locus (DYT13) within a 22 cM interval on the short arm of chromosome 1, with a maximum lod score of 3.44 between the disease and marker D1S2667.

Localization of a novel locus for autosomal recessive early-onset parkinsonism, PARK6, on human chromosome 1p35-p36.

The cause of Parkinson disease (PD) is still unknown, but genetic factors have recently been implicated in the etiology of the disease. So far, four loci responsible for autosomal dominant PD have been identified. Autosomal recessive juvenile parkinsonism (ARJP) is a clinically and genetically distinct entity; typical PD features are associated with early onset, sustained response to levodopa, and early occurrence of levodopa-induced dyskinesias, which are often severe. To date, only one ARJP gene, Parkin, has been identified, and multiple mutations have been detected both in families with autosomal recessive parkinsonism and in sporadic cases. The Parkin-associated phenotype is broad, and some cases are indistinguishable from idiopathic PD. In > or = 50% of families with ARJP that have been analyzed, no mutations could be detected in the Parkin gene. We identified a large Sicilian family with four definitely affected members (the Marsala kindred). The phenotype was characterized by early-onset (range 32-48 years) parkinsonism, with slow progression and sustained response to levodopa. Linkage of the disease to the Parkin gene was excluded. A genomewide homozygosity screen was performed in the family. Linkage analysis and haplotype construction allowed identification of a single region of homozygosity shared by all the affected members, spanning 12.5 cM on the short arm of chromosome 1. This region contains a novel locus for autosomal recessive early-onset parkinsonism, PARK6. A maximum LOD score 4.01 at recombination fraction .00 was obtained for marker D1S199.

A five-base pair deletion in the sedlin gene causes spondyloepiphyseal dysplasia tarda in a six-generation Arkansas kindred.

A six-generation kindred from Arkansas with X-linked recessive spondyloepiphyseal dysplasia tarda (SEDT) was investigated by genetic linkage and mutation analysis. SEDT had been mapped on the X-chromosome (Xp22.2), and the clinical and radiographic evolution of this kindred had been published. Linkage analysis proved informative for all five polymorphic markers tested, and DXS987 and DXS16 co-segregated with the Arkansas kindred (peak logarithm of the odds scores, 3.54 and 3.36, respectively). Subsequently, dinucleotide deletion in a new gene designated "sedlin" was reported to cause SEDT in three families. In an affected man and obligate carrier woman in the Arkansas kindred, we found a 5-bp deletion in exon 5 of sedlin. The defect causes a frameshift, resulting in eight missense amino acids and premature termination. The 5-bp deletion was then demonstrated to segregate with SEDT in the four living generations, including eight affected males and nine obligate carrier females. Furthermore, the deletion was identified in four females who potentially were heterozygous carriers for SEDT. The mutation was not detected in the two young sons of the consultand (believed to be a carrier because of her subtle radiographic skeletal changes and then shown to have the deletion), but they were too young for x-ray diagnosis Identification of a defect in sedlin in this SEDT kindred enables carrier detection and presymptomatic diagnosis and reveals an important role for this gene in postnatal endochondral bone formation.

A second paroxysmal kinesigenic choreoathetosis locus (EKD2) mapping on 16q13-q22.1 indicates a family of genes which give rise to paroxysmal disorders on human chromosome 16.

Paroxysmal kinesigenic choreoathetosis (PKC) is a rare paroxysmal movement disorder characterized by recurrent and brief attacks of choreiform or dystonic movements triggered or exacerbated by sudden voluntary movements. Some patients with PKC also have a history of infantile afebrile convulsions. PKC can be sporadic, or familial with autosomal dominant inheritance. PKC has been mapped to the pericentromeric region of human chromosome 16 in several Japanese families and in an African-American family, to regions which overlap by 9.8 cM (centiMorgan). Both regions overlap by 3.4 cM with a region containing a gene responsible for 'infantile convulsions and paroxysmal choreoathetosis' (ICCA). We have identified a second PKC locus (EKD2) on the long arm of chromosome 16 in a large Indian family with PKC. A maximum two-point LOD score of 3.66 (recombination fraction = 0.00, penetrance = 0.80) was obtained between PKC and D16S419. Haplotype and recombinant analysis localized EKD2 to a 15.8 cM region between D16S685 and D16S503. This region does not overlap with that identified in Japanese families, or with the ICCA locus. These results exclude one locus on chromosome 16 which causes both the ICCA and PKC syndromes; this suggests that there may be a cluster of genes on human chromosome 16 which lead to paroxysmal disorders.

Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking.

Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy with serious consequences for the mother and fetus. Two pedigrees have been reported with ICP in the mothers of children with a subtype of autosomal recessive progressive familial intrahepatic cholestasis (PFIC) with raised serum gamma-glutamyl transpeptidase (gamma-GT). Affected children have homozygous mutations in the MDR3 gene (also called ABCB4 ), and heterozygous mothers have ICP. More frequently, however, ICP occurs in women with no known family history of PFIC and the genetic basis of this disorder is unknown. We investigated eight women with ICP and raised serum gamma-GT, but with no known family history of PFIC. DNA sequence analysis revealed a C to A transversion in codon 546 in exon 14 of MDR3 in one patient, which results in the missense substitution of the wild-type alanine with an aspartic acid. We performed functional studies of this mutation introduced into MDR1, a closely related homologue of MDR3. Fluorescence activated cell sorting (FACS) and western analysis indicated that this missense mutation causes disruption of protein trafficking with a subsequent lack of functional protein at the cell surface. The demonstration of a heterozygous missense mutation in the MDR3 gene in a patient with ICP with no known family history of PFIC, analysed by functional studies, is a novel finding. This shows that MDR3 mutations are responsible for the additional phenotype of ICP in a subgroup of women with raised gamma-GT.

Autosomal dominant cerebellar ataxia type III: linkage in a large British family to a 7.6-cM region on chromosome 15q14-21.3.

Autosomal dominant cerebellar ataxia type III (ADCA III) is a relatively benign, late-onset, slowly progressive neurological disorder characterized by an uncomplicated cerebellar syndrome. Three loci have been identified: a moderately expanded CAG trinucleotide repeat in the SCA 6 gene, the SCA 5 locus on chromosome 11, and a third locus on chromosome 22 (SCA 10). We have identified two British families in which affected individuals do not have the SCA 6 expansion and in which the disease is not linked to SCA 5 or SCA 10. Both families exhibit the typical phenotype of ADCA III. Using a genomewide searching strategy in one of these families, we have linked the disease phenotype to marker D15S1039. Construction of haplotypes has defined a 7.6-cM interval between the flanking markers D15S146 and D15S1016, thereby assigning another ADCA III locus to the proximal long-arm of chromosome 15 (SCA 11). We excluded linkage of the disease phenotype to this region in the second family. These results indicate the presence of two additional ADCA III loci and more clearly define the genetic heterogeneity of ADCA III.

Mapping the gene causing hereditary primary hyperparathyroidism in a Portuguese kindred to chromosome 1q22-q31.

A Portuguese kindred with autosomal dominant isolated primary hyperparathyroidism (HPT) that was associated with parathyroid adenomas and carcinomas was investigated with the aim of determining the chromosomal location of this gene, designated HPTPort. Leukocyte DNA from 9 affected and 16 unaffected members and 7 parathyroid tumors from 4 patients was used in comparative genomic hybridization (CGH), tumor loss of heterozygosity (LOH), and family linkage studies. The CGH studies revealed abnormalities of chromosomes 1 and 13, and the results of LOH studies were consistent with the involvements of tumor suppressor genes from these regions. Family segregation studies mapped HPTPort to chromosome 1q22-q31 by establishing linkage with eight loci (D1S254, D1S222, D1S202, D1S238, D1S428, D1S2877, D1S422, and D1S412) (peak two-point LOD scores = 3. 46-5.14 at 0% recombination), and defined the location of HPT Port to a 21 cM region flanked centromerically by D1S215 and telomerically by D1S306. Thus, HPTPort has been mapped to chromosome 1q22-q31, and a characterization of this gene will help to elucidate further the mechanisms that are involved in the development of parathyroid tumors.

Localization of familial benign hypercalcemia, Oklahoma variant (FBHOk), to chromosome 19q13.

Calcium homeostasis by the kidneys and parathyroids is mediated by the calcium-sensing receptor (CaSR), which is located on 3q21-q24 and belongs to family C of the superfamily of G-protein coupled receptors that includes those for metabotropic glutamate, certain pheromones, and gamma-amino butyric acid (GABA-B). Inactivating CaSR mutations result in familial benign hypercalcemia (FBH), or familial hypocalciuric hypercalcemia (FHH), whereas activating mutations result in hypocalcemic hypercalciuria. However, not all FBH patients have CaSR mutations, which, together with the mapping of another FBH locus to 19p13.3, suggests that additional CaSRs or second messengers may be involved. These may be identified by positional cloning, and we therefore performed a genomewide search, using chromosome-specific sets of microsatellite polymorphisms, in an Oklahoma family with an FBH variant (FBHOk), for which linkage to 3q and 19p had been excluded. Linkage was established between FBHOk and eight chromosome 19q13 loci, with the highest LOD score, 6.67 (recombination fraction.00), obtained with D19S606. Recombinants further mapped FBHOk to a <12-cM interval flanked by D19S908 and D19S866. The calmodulin III gene is located within this interval, and DNA sequence analysis of the coding region, the 5' UTR, and part of the promoter region in an individual affected with FBHOk did not detect any abnormalities, thereby indicating that this gene is unlikely to be implicated in the etiology of FBHOk. This mapping of FBHOk to chromosome 19q13 will facilitate the identification of another CaSR or a mediator of calcium homeostasis.

Localisation of X linked recessive idiopathic hypoparathyroidism to a 1.5 Mb region on Xq26-q27.

X linked recessive idiopathic hypoparathyroidism (HPT) has been observed in two kindreds from Missouri, USA. Affected subjects, who are males, suffer from infantile onset of epilepsy and hypocalcaemia, which appears to be the result of an isolated congenital defect of parathyroid gland development; females are not affected and are normocalcaemic. The gene causing HPT has been previously mapped to a 7 cM interval, flanked centromerically by F9 and telomerically by DXS98, in Xq26-q27, and an analysis of mitochondrial DNA has established a common ancestry for these two kindreds. In order to define further the map location of HPT and thereby facilitate its isolation, we have undertaken linkage studies using polymorphic loci whose order has been established as Xcen - DXS1001 - DXS294 - DXS102 - F9 - DXS1232 - DXS984 - CDR1 - DXS105 - DXS1205 - DXS1227 - DXS98 - DXS52 - Xqter, within this region. Our results established linkage (lod score > 3) between HPT and eight of these 12 loci and indicated that the most likely location of HPT was within a 1.5 Mb interval flanked centromerically by F9 and telomerically by DXS984. Thus, the results of this study have helped to refine the map location of HPT, and this will facilitate the identification of this putative developmental gene and its role in the embryological formation of the parathyroids.

Mutational analysis of PHEX gene in X-linked hypophosphatemia.

Hypophosphatemic rickets is commonly an X-linked dominant disorder (XLH or HYP) associated with a renal tubular defect in phosphate transport and bone deformities. The XLH gene, referred to as PHEX, or formerly as PEX (phosphate regulating gene with homologies to endopeptidases on the X-chromosome), encodes a 749-amino acid protein that putatively consists of an intracellular, transmembrane, and extracellular domain. PHEX mutations have been observed in XLH patients, and we have undertaken studies to characterize such mutations in 46 unrelated XLH kindreds and 22 unrelated patients with nonfamilial XLH by single stranded conformational polymorphism and DNA sequence analysis. We identified 31 mutations (7 nonsense, 6 deletions, 2 deletional insertions, 1 duplication, 2 insertions, 4 splice site, 8 missense, and 1 within the 5' untranslated region), of which 30 were scattered throughout the putative extracellular domain, together with 6 polymorphisms that had heterozygosity frequencies ranging from less than 1% to 43%. Single stranded conformational polymorphism was found to detect more than 60% of these mutations. Over 20% of the mutations were observed in nonfamilial XLH patients, who represented de novo occurrences of PHEX mutations. The unique point mutation (a-->g) of the 5'untranslated region together with the other mutations indicates that the dominant XLH phenotype is unlikely to be explained by haplo-insufficiency or a dominant negative effect.