Hereditary haemochromatosis is an autosomal recessive disorder of iron regulation that results in abnormal intestinal iron absorption with progressive iron overloading of parenchymal cells. Two specific, single point mutations of the HFE gene (C282Y and H63D) have been described in haemochromatosis patients. Epidemiological studies have revealed a strict association between hereditary haemochromatosis and C282Y homozygosis or C282Y/H63D compound heterozygosis, suggesting that these mutations may provide a useful tool for diagnosis. However, recent investigations from southern Europe have reported lower allelic frequencies of the C282Y mutation among haemochromatosis patients, apparently depending on the geographical area of the population analysed. To assess the predictive value of the detection of the C282Y and H63D HFE mutations in our geographical area, we have evaluated their occurrence in 46 haemochromatosis patients from southern Italy. We found that only 19.6% of our patients were homozygous for the C282Y mutation and 21.7% were compound C282Y/H63D heterozygotes. Among the remaining 59%, approximately 40% did not display any of the known HFE mutations. We conclude that, in southern Italy, another genetic determinant/s must be responsible for many haemochromatosis cases and that a genetic screening for the C282Y and H63D HFE mutations is not sufficient for hereditary haemochromatosis diagnosis.

We have developed and clinically tested a rapid and largely automated procedure to detect mutations in the coding region of a gene of interest. Our method relies on the automated sequencing of the complete cDNA, followed by an advanced mutation search-and-verification routine using an integrated set of computer analysis tools. We have applied our automated procedure to the diagnosis of familial hypercholesterolemia (FH) in 52 unrelated FH families, by sequencing the whole cDNA coding region of the LDLR gene. Here we report the procedures and performance of our method in the identification of the most common types of LDLR mutations: short deletions or insertions and point mutations. Our method can provide a standard procedure for the 'overnight' unequivocal identification of mutations in those genetic diseases where several different mutations, none clearly prominent, may affect a given gene.

We screened a group of patients from southern Italy with clinically diagnosed familial hypercholesterolemia (FH) for mutations of the LDL receptor (LDLR) gene. RNA from each proband was analysed by RT-PCR followed by complete cDNA sequencing. Among 51 unrelated FH families we detected 17 mutations affecting the coding region of the LDLR gene. Five of these mutations, designated R395P, L783fsinsG, IVS15-3C>A, IVS3+5G>A, and 1698-1704delCACCCTAinsGCCCAAT (ITL545MPN), have not yet been reported in the literature. Interestingly, the novel IVS15-3C>A splicing mutation was detected in 20% of our unrelated FH families, suggesting an unusually high prevalence in our local population. Hum Mutat 17:433, 2001.

Genotypic abnormalities of the renin-angiotensin system have been suggested as a risk factor for the development of microangiopathic complications in diabetic patients. We studied the relationship of either an insertion-deletion polymorphism in the angiotensin-converting enzyme (ACE) gene and the M235T and T174M variant polymorphisms of the angiotensinogen (AGT) gene in non—insulin-dependent diabetes mellitus (NIDDM) patients and its relationship with cardiovascular complications. A total of 193 NIDDM patients (89 men and 104 women aged 59.2 ± 10.0 years; diabetes duration, 13.2 ± 6.2 years) and 90 control subjects (42 men and 48 women aged 45.4 ± 12.6 years) were recruited for the association study. Distribution of the genotype or allelic frequencies for all the studied polymorphisms did not differ significantly between controls and NIDDM patients. ACE and AGT genes did not display any difference in clinical or metabolic parameters according to each gene's genotype for either the control or the NIDDM group, For evaluation of nephropathy and retinopathy, NIDDM patients were matched with subjects not having microangiopathic complications. Thus, a total of 60 patients had diabetic nephropathy and were compared with 100 patients with normoalbuminuria. Sixty-eight NIDDM patients had diabetic retinopathy, and 92 patients presented no signs of retinopathy. There were no differences in genotypic or allelic distribution between NIDDM patients for either the presence or absence of retinopathy or nephropathy. We conclude that the ACE and AGT polymorphisms do not contribute to the genetic susceptibility to diabetic nephropathy and retinopathy in a caucasian Mediterranean population.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25 000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.