Mutation detection in the Tyrosine Hydroxylase gene (TH) was performed in patients from two families. DNA sequencing revealed the presence of four novel missense mutations (exon 9 and 14 in family A, exon 8 and 9 in family B); the mutations were confirmed with restriction enzyme analysis, and did not occur in control alleles. Three mutations are in the catalytic domain of the enzyme and one may disturb tetramerization. At the moment, all patients are in the fourth decade of life. For more than 30 years they have been able to live a normal life with low-dose L-DOPA medication.

Earlier analysis of the Italian population showed patterns of genetic differentiation that were interpreted as being the result of population settlements going back to pre-Roman times. DNA disease mutations may be a powerful tool in further testing this hypothesis since the analysis of diseased individuals can detect variants too rare to be resolved in normal individuals. We present data on the relative frequencies of 60 cystic fibrosis (CF) mutations in Italy and the geographical distribution of the 12 most frequent CF mutations screened in 3492 CF chromosomes originating in 13 Italian regions. The 12 most frequent mutations characterize about 73% of the Italian CF chromosomes. The most common mutation, ΔF508, has an average frequency of 51%, followed by N1303K and G542X, both with average frequencies around 5%. Multivariate analyses show that the relative frequencies of CF mutations are heterogeneous among Italian regions, and that this heterogeneity is weakly correlated with the geographical pattern of non-DNA ‘classical’ genetic markers. The northern regions are well differentiated from the central-southern regions and within the former group the western and eastern regions are remarkably distinct. Moreover, Sardinia shows the presence of mutation T338I, which seems absent in any other European CF chromosome. The north-western regions of Italy, characterized by the mutation 1717-1G→A, were under Celtic influence, while the north-east regions, characterized by the mutations R1162X, 2183AA→G and 711­5G→A, were under the influence of the Venetic culture.

As part of the 1990/1991 Pakistan Demographic and Health Survey, data were collected on the outcome of 26408 births to 6611 women, with mortality rates investigated at specific age intervals during the first 5 years of life. Bivariate and multivariate logistic regression analyses were employed to examine the comparative roles of consanguineous marriage and a number of demographic and socioeconomic factors, including the sex of the child, maternal age, maternal education, birth interval and birth order, as determinants of early death. The results indicate that, even after controlling for these non-genetic variables, inbreeding at the level of first cousin exerted a significant adverse effect on survival in four of the five age intervals examined, neonatal, post-neonatal, infant and under 5 years.

The Klippel–Trenaunay–Weber syndrome (KTWS) is generally thought to occur sporadically, following a somatic mutation model. However, in some cases, clinical manifestations of the syndrome have been found in family members, suggesting an autosomal dominant inheritance. Here we present an epidemiological analysis of a consecutive series of cases with KTWS identified in the Spanish Collaborative Study of Congenital Malformations (ECEMC). We found an increase in parental age and in the number of pregnancies, as well as familial occurrence of haemangiomas. These observations suggest a genetic contribution to the occurrence of KTWS.

The original transmission disequilibrium test (TDT), was introduced to test for linkage between a marker and a disease-susceptibility locus (Spielman et al. 1993). Allison (1997) extended the TDT procedure to quantitative traits. Allison's test, however, is restrictive in that it requires family trios consisting of one heterozygous parent, one homozygous parent and one child, and considers only the situation where the marker locus is analogous to the quantitative trait locus itself. In this paper, we propose, investigate and apply a general TDT for quantitative traits that permits more than one child per family, does not require only one parent to be heterozygous, and allows for the fact that the various alleles at the marker and trait loci may be at varying degree of linkage disequilibrium. We also show that this TDT for quantitative traits is still a valid test of linkage in the presence of population substructure. To provide guidelines for study design, we develop analytic formulae for calculation of the power of the TDT for mapping quantitative trait loci and investigate the impact of various factors on the power. Power calculations show that the proposed TDT for quantitative traits is more powerful than Allison's basic test statistic and the extreme discordant sib pair linkage method. The proposed TDT statistic for quantitative traits is applied to systolic blood pressure variation in the Rochester Family Heart Study using an extremely discordant sibling pair design.

Single nucleotide polymorphisms (SNPs) are very common throughout the genome and hence are potentially valuable for mapping disease susceptibility loci by detecting association between SNP markers and disease. However as SNPs are biallelic they may have relatively little power in association studies compared with the information that would be obtainable if marker haplotypes were available and could be used efficiently. Modelling the evolutionary events leading to linkage disequilibrium is very complex and many methods that seek to use information from multiple markers simultaneously need to make simplifying assumptions and may only be applicable when marker haplotypes, rather than genotypes, are available for analysis. We explore the properties of a simple application of a standard artificial neural network to this problem. The pattern-recognition properties of the network are used in the hope that marker haplotypes implicit in the genotypes will differ between cases and controls in a way which will lead to the network being able to classify the subjects correctly, according to their marker genotype. This method makes no assumptions at all regarding population history or the marker map, and can be applied to genotypes, as would be available from a simple case-control sample, without any need to determine haplotypes. Through application to data simulated under a very wide range of assumptions we show that such an analysis produces a useful augmentation in power above that which would be achieved by testing each marker individually, in particular when more than one mutation has occurred in a disease gene at different points in evolution. The application of neural networks to such problems shows considerable promise and further work could usefully be directed towards optimising the design and implementation of such networks.

We report a branch site mutation in the gene of the enzyme tyrosine hydroxylase (TH): a −24t > a substitution two bases upstream of the adenosine in the branchpoint sequence (BPS) of intron 11. As normal lariat formation is therefore prevented, alternative splicing takes place: use of the BPS of intron 12 results in skipping of exon 12, whereas the use of a cryptic branch site in intron 11 leads to partial retention of this intron in the mRNA. This leads in both cases to an aberrant protein product. In the one case, skipping of exon 12 results in the absence of 32 amino acids. In the other, retention of 36 nucleotides of intron 11 in the mRNA results in the incorporation of twelve additional amino acids. The functional consequences of this mutation for the patient, who is also heterozygous for another previously identified mutation, become apparent in a severe clinical phenotype.

Bone mineral content (BMC) and/or bone mineral density (BMD, i.e. BMC scaled by bone size) are major determinants for osteoporosis, which is a serious health problem. The major determinant of variation in BMD/BMC is genetic. The few studies now available are inconsistent in the identification and/or even in the existence of major gene(s) for BMD/BMC. In 51 human pedigrees with 941 individuals (526 measured for phenotypes) identified via probands with extreme BMD values, we performed complex segregation analyses to test the existence of a genetic locus with a major effect on BMD/BMC variation. We analyzed BMD and BMC at the spine, hip and wrist jointly by employing, as the study phenotype, factor scores (FS) of the principle component that explains ∼75% of the total BMD/BMC variation at the three sites. The results indicate that a major gene exists with a codominant effect that is responsible for ∼16% of the FS variation when adjusted for significant effects of sex, body weight and age. A significant genotype-×-sex-×-age interaction was found, which may explain ∼14% of the FS variation after adjusting for body weight. Testing of various models did not provide support for shared familial environmental effects but suggested the existence of residual polygenic effects, which may explain ∼50% of the FS variation when adjusting for sex, body weight and age. This study indicates a promising aspect of studies to identify a major gene for BMD/BMC variation in our pedigrees identified via extreme probands.

FRAXE fragile site associated mental retardation remains unique among X-linked mental retardation phenotypes due to its very mild to borderline nature (50 < IQ < 85). It is the most prevalent form of non-specific X-linked mental retardation so far delineated, with an estimated incidence of at least 1/50–100000 males, and with more than 50 families known worldwide. The FRAXE site is within, or immediately adjacent to, the 5′ untranslated region of the FMR2 gene. Hyperexpansion of the FRAXE CCG repeat silences transcription of the gene. The structure of FMR2 has been characterized, but its function remains unknown. Gene localizations for numerous (> 75) large families with non-specific X-linked mental retardation (MRX) have been determined so far. Recently the molecular basis for some of them has been unravelled by identification of the responsible genes, which participate in complex common signalling pathways. This review summarises the new data on FRAXE associated mental retardation and the FMR2 gene in the light of the recent discoveries of new genes responsible for other forms of non-specific X-linked mental retardation.

We performed a comprehensive analysis for mutations in the TSC1 gene using Southern blot analysis, and SSCP and heteroduplex analysis of amplified exons in 13 families with genetic linkage to the TSC1 region, 22 small families without linkage information, and 126 sporadic patients. 17 unique mutations were identified in 21 patients. Mutations were found in 7/13 (54%) TSC1-linked families, 1/22 (5%) small families without linkage, and 13 of 126 (10%) sporadic cases. The mutations were all chain-terminating, with 14 small deletions, 1 small insertion, and 6 nonsense mutations. In families with mutations, all individuals carrying a mutation met formal diagnostic criteria for TSC, apart from a 3-year-old girl who had inherited a deletion mutation, and who had no seizures, normal intelligence, normal abdominal ultrasound, and hypomelanotic macules only on physical exam. We assessed the incidence and severity of mental retardation in the 13 sporadic patients with TSC1 mutations versus the entire sporadic cohort, and found no significant difference. The observations indicate that TSC1 mutations are all inactivating, suggest that TSC1 disease occurs in only 15–20% of the sporadic TSC population, and demonstrate that presymptomatic TSC does occur.

The entire coding region of the TSC1 gene has been screened for mutations in 79 unrelated patients with tuberous sclerosis. Causative mutations have been found in 27 of these patients and five other variations in the gene have been identified. 26 of the mutations are predicted to cause premature truncation of the protein product of the gene and one mutation is in a splice site. The mutation screen has revealed that TSC1 mutations are rarer in sporadic tuberous sclerosis patients than in familial cases. We have also found that the only previously described case of non-penetrance can no longer be described as such, and that a single ungual fibroma is not necessarily diagnostic of tuberous sclerosis, important findings for the genetic counselling of tuberous sclerosis patients.

It was investigated whether radiographic osteoarthritis (ROA) is associated with specific haplotypes of the COL2A1 gene. Radiographs of knees, hips, hands, and spine were scored for the presence of ROA in subjects of 55–70 years from a population-based cohort study, the Rotterdam study. Cases had ROA in 3 or more joint groups; controls, from the same population, had ROA in less than 3 joint groups. Allele frequencies of 3 dimorphisms (HaeIII, HindIII, MaeII) and a VNTR polymorphism of the COL2A1 gene were determined. The VNTR allele 14R2 and the HindIII polymorphism showed a significant association. Haplotype analysis of the HaeIII, HindIII and VNTR polymorphisms showed that a specific haplotype (1-2-14R2) is strongly associated with ROA in 3 or more joint groups (OR = 5.3, 95% CI 2.3–12.7). Our results suggest that a specific haplotype of the COL2A1 locus may predispose to generalised ROA.

The hypervariable segment I of the control region of the mtDNA was sequenced in 45 unrelated individuals from Bioko and 50 from São Tomé, two islands in the Gulf of Guinea that have had very different settlement patterns: Bioko was colonized around 10000 BP, while São Tomé was first settled by the Portuguese, who brought African slaves to the island. Two different patterns of sequence variation are evident and are also clearly a consequence of their very different demographic histories. The Bubi present a low genetic diversity and it is likely that the island was colonized by a small number of individuals with small later migration. São Tomeans might be considered a subset of a mainland African population relocated to the island. They present high genetic diversity with a high number of sequences being shared with many continental populations. This study, with knowledge of the population history in island populations, strengthens the genetic approach to unravel past demographic events.

We typed 1801 males from 55 locations for the Y-specific binary markers YAP, DYZ3, SRY10831 and the (CA)n microsatellites YCAII and DYS413. Phylogenetic relationships of chromosomes with the same binary haplotype were condensed in seven large one-step networks, which accounted for 95% of all chromosomes. Their coalescence ages were estimated based on microsatellite diversity. The three largest and oldest networks undergo sharp frequency changes in three areas. The more recent network 3.1A clearly discriminates between Western and Eastern European populations. Pairwise Fst showed an overall increment with increasing geographic distance but with a slope greatly reduced when compared to previous reports. By sectioning the entire data set according to geographic and linguistic criteria, we found higher Fst-on-distance slopes within Europe than in West Asia or across the two continents.

Hereditary haemochromatosis is a common inherited disorder leading to excessive accumulation of iron in various organs. Two missense substitutions at the HFE-gene have recently been associated with the disease, 187C G and 845G → A (mutations H63D and C282Y, respectively). We present a simple, rapid PCR–SSCP multiplex screening method allowing the simultaneous detection of both substitutions. Furthermore, testing the method on 420 Danish blood donors revealed the presence of a hitherto undetected third substitution in 13 individuals. The new substitution, a 193A → T transversion, affects codon 65 changing the code for serine to that of cysteine (S65C). It may thus have functional consequences for the HLA class protein encoded by the HFE-gene. The allele frequencies observed were: H63D 14.8%, C282Y 6.2% and S65C 1.5%, which for the two former alleles are in agreement with frequencies reported for other North European population samples.

We genotyped 64 dinucleotide microsatellite repeats in individuals from populations that represent all inhabited continents. Microsatellite summary statistics are reported for these data, as well as for a data set that includes 28 out of 30 loci studied by Bowcock et al. (1994) in the same individuals. For both data sets, diversity statistics such as heterozygosity, number of alleles per locus, and number of private alleles per locus produced the highest values in Africans, intermediate values in Europeans and Asians, and low values in Americans. Evolutionary trees of populations based on genetic distances separated groups from different continents. Corresponding trees were topologically similar for the two data sets, with the exception that the (δμ)2 genetic distance reliably distinguished groups from different continents for the larger data set, but not for the smaller one. Consistent with our results from diversity statistics and from evolutionary trees, population growth statistics Sk and β, which seem particularly useful for indicating recent and ancient population size changes, confirm a model of human evolution in which human populations expand in size and through space following the departure of a small group from Africa.

Decreased function of the melanocortin-4 receptor (MC4R) was reported to cause late-onset obesity and insulin resistance in rodents. Thus mutations in the MC4R gene drew strong attention as a possible cause of obesity and diabetes. We screened for mutations in the MC4R gene in extremely obese [body mass index (BMI) [ges ] 35 kg/m2] Japanese with diabetes by direct sequencing. A heterozygous mutation (V103I) was detected in one case (2.0 %), however the frequency was not significantly different from that in non-obese (BMI [les ] 24 kg/m2) and non-diabetic subjects (2.7 %). No other mutations were detected. These results suggest that mutations including V103I in the MC4R gene are not a major cause of obesity or diabetes in Japanese.

We defined the Y-chromosome haplotypes on the basis of six polymorphic sites: an Alu-element insertion (YAP), a single-base change (C→T at DYS199), one trinucleotide repeat (DYS392) and three tetranucleotide repeats (DYS393, DYS390 and DYS19). Among 140 Y chromosomes from Whites, Blacks, Japanese and Amerindians we identified 67 different haplotypes, the majority of them population-specific; only seven haplotypes were shared by three different racial groups, mostly owing to admixture. Overall, three main lineages can be defined on the basis of the YAP/DYS199/DYS392 markers: (a) a predominant /−/C/10/13/22 (or) 23/ lineage, observed among all racial groups; (b) a/+/C/ lineage which predominates among Blacks (comprising mainly the sublineage /+/C/10/13/), although it is eventually found among Japanese and Whites; and (c) a /−/T/ lineage observed only among Amerindians (comprising mainly the sublineage /−/T/13/13/). The decreasing haplotype diversity of the three lineages agrees with the idea that the first is the most ancient, while the last is the more recent. The data also indicate that the YAP insertion occurred in a /−/C/10/13/ chromosome and the C→T mutation occurred in a /−/C/13/13/ chromosome. Finally, the data suggest that at least two Y-chromosome lineages (/−/C/13/ and /−/T/13/) contributed to the early peopling of the Americas, and supports the hypothesis that /−/T/13/ could be derived from /−/C/13/ and that both haplotypes could be present in the ancestral populations that peopled the continent.

An estimated 5–10% of all breast and ovarian cancers are due to an inherited predisposition, representing a rather large number of patients. In Spain 1/13–1/14 women will be diagnosed with breast cancer during their lifetime. Two major breast cancer genes, BRCA1 and BRCA2, have been identified. To date, several hundred pathogenic mutations in these two genes have been published or reported to the Breast Cancer Information Core, BIC database (http://www.nhgri.nih.gov/Intramural_research_Lab  transfer/Bic/index.html). In the present study, 30 Spanish breast and breast/ovarian cancer families (29 from Galicia, NW Spain, and 1 from Catalonia, NE Spain) were screened for mutations in the BRCA1 and BRCA2 genes. The analysis of these genes was carried out by SSCP for shorter exons and direct sequencing in the case of longer ones. Mutations were found in 8 of the 30 families studied (26.66%). It is important to note that all mutations were detected within the BRCA1 gene: 330 A>G, 910_913delGTTC, 2121 C>T, 3958_3962delCTCAGinsAGGC, and 5530 T>A. The BRCA1 330 A>G mutation was found in four unrelated families and accounted for 50% of all identified mutations.

Linkage disequilibrium is an important tool both at the end stages of positional cloning studies to map genes of particular interest and in reconstruction of population histories. With advances in molecular biology, complex genetic systems involving multiple highly polymorphic markers are becoming more commonly used to study linkage disequilibrium. These systems contain much more genetic information than simple two marker systems. In this article, we introduce a measure to summarize the overall deviation from random association and propose a permutation-based estimate for this measure. The performance of the proposed estimation procedure is studied through simulations. The methods proposed in this paper are then applied to population data at the dopamine D2 receptor locus (DRD2) and at the homeobox B (HOXB) gene cluster.