Population-wide evaluation of disease manifestation in relation to molecular genotype in steroid 21-hydroxylase (CYP21) deficiency: good correlation in a well defined population.

We report a population-wide analysis of all patients with 21-hydroxylase deficiency (21-OHD) found in Finland, a country with a genetically well defined population, in which the effects of other genetic and environmental factors on the phenotype can be expected to be low. In total, 120 patients were identified, and their clinical status was evaluated. Blood samples for CYP21 genotype determination could be obtained from 78 (65%) patients, and their phenotypes were compared with their genotypes. In general, the severity of gene defects correlated well with clinical expression. All patients carrying mutations with the most drastic effects on enzymatic activity had the salt-wasting form of 21-OHD. The I2 splice mutation, which in some reports has been connected with clinical variation, was constantly associated with severe mineralocorticoid deficiency. However, patients with I172N as the determining mutation expressed a wide spectrum of phenotypes; the variation could not be attributed to additional mutations. Although genetically affected males with the nonclassical form had not been clinically diagnosed, our study suggests that nonclassical 21-OHD is substantially more rare in Finland than elsewhere, as indicated by both clinical evaluation and mutational screening.

Tracing past population migrations: genealogy of steroid 21-hydroxylase (CYP21) gene mutations in Finland.

The genealogic origin of steroid 21-hydroxylase gene (CYP21) mutations and associated haplotypes was determined in 74 unrelated Finnish families with CYP21 deficiency (congenital adrenal hyperplasia, CAH). These families account for two thirds (85/119) of all diagnosed patients of Finnish descent found in this country. We recently demonstrated that multiple founder mutations each associated with a particular haplotype can be found in Finland. Interestingly, some of the haplotypes were identical to those observed in various European populations, whereas others have not been described elsewhere, indicating a local and perhaps a more recent origin. In the present report we show that each of the major founder haplotypes originates from a particular geographic region of Finland. Thus many local genetic isolates are to be expected in Finland. Our finding is in a clear contrast to the genetic diseases known as the 'Finnish disease heritage', in which only one mutation usually predominates. Some of the CYP21 haplotypes proved very informative for analysis of the history of the Finnish population. For example, the origin of one frequent haplotype was shown to cluster in a region assumed by archaeological data to be a major site of immigration by settlers of either Scandinavian or Baltic origin during the first centuries AD. As this haplotype is frequent in many European patient populations, we provide independent genetic evidence of this Iron Age immigration. On the other hand, another frequent haplotype found solely in Finland reflects a more recent (post 15th century) settlement expansion. Consequently, well characterised and sufficiently frequent autosomal gene markers can provide useful information on migrations both between and within populations.

Single founder mutation (W380G) in type II protein C deficiency in Finland.

The present study investigated the genetic basis for type II protein C deficiency in Finland, where this form has an unusually high incidence. We demonstrated that, first, a single novel mutation W380G in the protein C gene (PROC) explained 25/26 index patients, estimated to represent two thirds of all families with type II deficiency in Finland. Second, extended chromosomal conservation, i.e. a specific haplotype, around the W380G mutation was indicated in unrelated patients. Third, a local geographical origin for the W380G mutation was suggested by genealogical data. These results are in contrast to the heterogeneity in type II protein C deficiency elsewhere, but closely parallel disorders of the Finnish disease heritage. The high frequency of the type II disease can be explained by founder effect and subsequent enrichment of a single mutation in Finland. The present study also provided a simple means for genetic diagnosis of this disease and the genetic test can be included in the routine screenings in this population.

A rare neutral polymorphism in 21-hydroxylase genes as HLA haplotype marker. Evidence for strong founder effect in the Finnish population.

The usefulness of rare neutral gene polymorphisms as an HLA haplotype marker and as a probe for founder effect in small populations was tested by determining the frequency and MHC associations of an NcoI polymorphism in the P450c21 genes in the Finnish population. In the general population, 13% (9 of 70) of samples had the NcoI site. A very strong association with the HLA-B62, Bf*F, C4A*3, C4B*Q0, DRB1*13, DQA1*0103, DQB1*0603 alleles was observed. P450c21A and P450c21B gene-specific amplifications mapped the polymorphic site to both P450c21A pseudogene and P450c21B functional gene of this haplotype in all cases. The majority of haplotypes with the NcoI cutting site found in this population may thus have derived from a single ancestral haplotype. The HLA homozygous cell lines with the NcoI site showed heterogeneous HLA associations. Our results suggest that in small populations the variety of MHC haplotypes may be surprisingly low and rare polymorphisms can serve as informative markers.

Death in bathtub revisited with molecular genetics: a victim with suicidal traits and a LQTS gene mutation.

A 44-year-old woman with a medical history of mental disorders and previous suicidal behaviour was found in a bathtub and pronounced death few minutes later despite of resuscitation attempts. After police investigation and on the basis of autopsy findings, the death was classified as suicide drowning. Retrospective examination of clinical data revealed, a prolonged rate-corrected QT-interval (QTc: 468 ms) 3 months before death. Post-mortem (PM) DNA analysis disclosed KCNH2(FIN) mutation for the long-QT syndrome (LQTS). The value of PM molecular screening for LQTS is emphasised, especially for victims of putative drowning.

Mutation-haplotype analysis of steroid 21-hydroxylase (CYP21) deficiency in Finland. Implications for the population history of defective alleles.

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency is a common inherited defect of adrenal steroid hormone biosynthesis. Unusually for genetic disorders, the majority of mutations causing CAH apparently result from recombinations between the CYP21 gene encoding the 21-hydroxylase enzyme and the closely linked, highly homologous pseudogene CYP21P. The CYP21 and CYP21P genes are located in the major histocompatibility complex class III region on chromosome 6p21.3. We analyzed the mutations and recombination breakpoints in the CYP21 gene and determined the associated haplotypes in 51 unrelated Finnish families with CAH. They represent no less than half of all CYP21 deficiency patients in Finland. The results indicate the existence of multiple founder mutation-haplotype combinations in the population of Finnish CAH patients. The three most common haplotypes constituted half of all affected chromosomes; only one-sixth of the haplotypes represented single cases. Each of the common haplotypes was shown consistently to carry a typical CYP21 mutation and only in some cases was additional variation observed. Surprisingly, comparisons with previous published data revealed that several of the frequent mutation-haplotype combinations in Finland are in fact also found in many other populations of patients of European origin, thus suggesting that these haplotypes are of ancient origin. This is in clear contrast to many reports, including the present one, where a high frequency of de novo mutations in the CYP21 gene has been reported. In addition, two unique sequence aberrations in CYP21 (W302X and R356Q), not known to exist in the CYP21P pseudogene, were detected.

Novel mutations in the human CYP21 gene.

The great majority of genetic defects underlying steroid 21-hydroxylase deficiency appear to result from intergenic recombinations between the homologous CYP21 and CYP21P genes. For a minority, novel sporadic point mutations have been detected. De novo mutations in CYP21 have also been reported, but only a few studies have systematically screened their occurrence. We here describe a population-based patient sample in order to estimate the rate of single-family (i.e. sporadic) and de novo germline mutations in the human CYP21 locus. Among 76 Finnish families were observed three single-family mutations and two de novo mutations in CYP21. The rates obtained, approximately 5% and approximately 2% for novel and de novo mutations, respectively, indicate that they are not rare and that their occurrence should not be ignored in genetic diagnostics of this disorder.

An approach to mapping haplotype-specific recombination sites in human MHC class III.

Studies of the major histocompatibility complex (MHC) in mouse indicate that the recombination sites are not randomly distributed and their occurrence is haplotype-dependent. No data concerning haplotype-specific recombination sites in human are available due to the low number of informative families. To investigate haplotype-specific recombination sites in human MHC, we here describe an approach based on identification of recombinant haplotypes derived from one conserved haplotype at the population level. The recombination sites were mapped by comparing polymorphic markers between the recombinant and assumed original haplotypes. We tested this approach on the extended haplotype HLA A3; B47; Bf*F; C4A*1; C4B*Q0; DR7, which is most suitable for this analysis. First, it carries a number of rare markers, and second, the haplotype, albeit rare in the general population, is frequent in patients with 21-hydroxylase (21OH) defect. We observed recombinants derived from this haplotype in patients with 21OH defect. All these haplotypes had the centromeric part (from Bf to DR) identical to the original haplotype, but they differed in HLA A and B. We therefore assumed that they underwent recombinations in the segment that separates the Bf and HLA B genes. Polymorphic markers indicated that all break points mapped to two segments near the TNF locus. This approach makes possible the mapping of preferential recombination sites in different haplotypes.

Major histocompatibility complex (MHC)-linked microsatellite markers in a founder population.

The Finnish population is genetically relatively homogeneous and has a narrow gene pool as a result of founder effect followed by rapid population growth. We here demonstrate that microsatellite markers are highly informative tools for major histocompatibility complex (MHC) analysis in this population. First, no variation in 12 MHC-linked microsatellites could be observed in certain CYP21-deficient chromosomes, which as a result of founder effect most likely derived from common ancestors. Second, amongst 131 Finnish chromosomes, some, but not all, apparently HLA-identical chromosomes also carried identical microsatellites, suggesting that these loci could be applied for identification of haplotypes which have a relatively recent shared origins. Finally, when the microsatellites were studied between ethnically more distant individuals (Finnish vs. non-Finnish), who were matched for the HLA alleles, much more differences were observed. This showed that the similarity in microsatellites was population specific. The microsatellite typing can therefore be informative in fine mapping MHC-linked susceptibility genes and can help in matching bone marrow transplants in isolated populations. Linkage disequilibrium was found to be much higher in the MHC than in another region (5q31) of similar size, indicating that there may be particular mechanisms keeping the MHC haplotypes conserved.

A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction.

Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. A total of 11 missense mutations has previously been implicated in this enzyme deficiency. We describe two novel missense mutations, both affecting the same amino acid residue, Arg356. The two mutations, R356P and R356Q, were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells, and enzyme activity towards the two natural substrates, 17-hydroxyprogesterone and progesterone, was determined. The R356P mutant reduced enzyme activity to 0.15% towards both substrates, whereas the R356Q mutant exhibited 0.65% of normal activity towards 17-hydroxyprogesterone, and 1.1% of normal activity towards progesterone. These activities correspond to the degrees of disease manifestation of the patients in whom they were found. Arg356 is located in a region which recently has been implicated in redox partner interaction, by modelling the structure of two other members of the cytochrome P450 superfamily. Of the 11 previously described missense mutations, three affect arginine residues within this protein domain. With the addition of R356P and R356Q, there is a clear clustering of five mutations to three closely located basic amino acids. This supports the model in which this protein domain is involved in redox partner interaction, which takes places through electrostatic interactions between charged amino acid residues.

The natural history of an HLA haplotype and its recombinants.

The presence of haplotype-specific recombination sites can be determined by analyzing the conservation of extended haplotypes in the population. This approach considers all meioses in the history of the population and requires the presence of characteristic markers that easily allow the identification of the haplotype or of its recombined segments. The recombination breakpoint can then be mapped by looking for shared alleles between haplotypes selected through the specific marker/s. We identified a rare perfect tandem duplication of a 145 base pair segment in the LTA promoter, which tags a B60 (B60D) haplotype. The duplication was detected in 16/90 B60+ Europeans, while absent in 101 B60+ Orientals. The conservation of the class I end and the extreme variability of the class II end suggested that the present-day B60D haplotypes originated from an ancestral haplotype by recombination events centromeric to the duplicated sequence. Through a fine mapping using markers of the HLA central region a preferential recombination site was localized in the 60 kilobase interval between TNFd,e, and D6S273/K11 Amicrosatellite loci (i.e., between LST1 and BAT3 genes). This site behaves as a potent recombination enhancer leading to fragmentation in most of the extant B60D haplotypes and can be considered responsible for their "instability". In the relatively recently founded Finnish population, where the LST1/BAT3 interval recombination has probably not yet had the chance to occur, a founder effect can explain the presence of a rare DP (DPB1(*)1601) allele in most B60D haplotypes in this population.