Axenfeld-Rieger syndrome resulting from mutation of the FKHL7 gene on chromosome 6p25.

Mutations in the forkhead-like 7 (FKHL7) gene have been recently shown to cause juvenile glaucoma and anterior segment anomalies. We report on a three-generation family with Axenfeld-Rieger syndrome (ARS), harboring an alteration in the FKHL7 gene. Genetic linkage analyses excluded the ARS phenotype from chromosomes 4q25 and 13q14, the locations of the PITX2 and RIEG2 loci, respectively. Evidence of linkage was observed with markers at 6p25, near the FKHL7 gene. Direct sequencing of FKHL7 detected a C67T mutation that segregated with the ARS phenotype in this family, but was not detected in over 80 control chromosomes. This mutation is predicted to cause a nonsense mutation of the FKHL7 protein (Gln23Stop) upstream of the forkhead DNA-binding domain, and thus to generate a truncated FKHL7 protein product. This discovery broadly implicates FKHL7 in ocular, craniofacial, dental, and umbilical development.

The gamma-crystallins and human cataracts: a puzzle made clearer.

Despite the fact that cataracts constitute the leading cause of blindness worldwide, the mechanisms of lens opacification remain unclear. We recently mapped the aculeiform cataract to the gamma-crystallin locus (CRYG) on chromosome 2q33-35, and mutational analysis of the CRYG-genes cluster identified the aculeiform-cataract mutation in exon 2 of gamma-crystallin D (CRYGD). This mutation occurred in a highly conserved amino acid and could be associated with an impaired folding of CRYGD. During our study, we observed that the previously reported Coppock-like-cataract mutation, the first human cataract mutation, in the pseudogene CRYGE represented a polymorphism seen in 23% of our control population. Further analysis of the original Coppock-like-cataract family identified a missense mutation in a highly conserved segment of exon 2 of CRYGC. These mutations were not seen in a large control population. There is no direct evidence, to date, that up-regulation of a pseudogene causes cataracts. To our knowledge, these findings are the first evidence of an involvement of CRYGC and support the role of CRYGD in human cataract formation.

CEPH consortium map of chromosome 14.

Families from the linkage panel of Centre d'Etude du Polymorphisme Humain have been used to generate a linkage map containing 68 loci; 13 genes, 33 di- and 4 tetranucleotide repeats, one oligonucleotide ligation assay (OLA), and 17 RFLPs. This map integrates markers from several previous maps, and has undergone further error checking. 43 loci have been placed with odds of 1000:1 or greater, five with odds of 100:1, with an average interval of 3.5 cM. An additional 20 loci have been placed within defined intervals.

Physical and genetic mapping of the serpin gene cluster at 14q32.1: allelic association and a unique haplotype associated with alpha 1-antitrypsin deficiency.

The alpha 1-antitrypsin (PI) gene is part of a cluster of structurally related serine protease inhibitor genes localized at chromosome 14q32.1, a cluster that includes the alpha 1-antichymotrypsin (AACT), protein C inhibitor (PCI), and corticosteroid-binding globulin (CBG) genes and the alpha 1-antitrypsin-like pseudogene (PIL). The order of the genes is refined here by genetic mapping using simple tandem repeat polymorphisms (STRPs) and by physical mapping in YACs. The order of the genes is (centromere)-CBG-PIL-PI-PCI-AACT-(telomere). Analysis of DNA haplotypes comprising STRP and RFLP markers in the serpin genes reveals considerable allelic association throughout the cluster. Furthermore, the common alpha 1-antitrypsin deficiency allele, PI*Z, has a unique DNA haplotype at the CBG, PIL, and PI loci, which extends over 60 kb in 97% of cases and in 44% of cases includes the PCI and AACT loci. This unique haplotype will be of use in examining a number of other diseases, particularly those with an inflammatory component, thought to be associated with alpha 1-antitrypsin deficiency or partial deficiency.

Regional localization of loci on chromosome 14 using somatic cell hybrids.

We have used a panel of human x rodent somatic cell hybrids containing translocations involving chromosome 14 to regionally localize 17 genes and 5 random segments previously mapped to chromosome 14. Each hybrid cell line contains a specific fragment of chromosome 14, with breakpoints at 14q11.2, 14q21, 14q22, 14q24.3 or in two different regions of 14q32.1. The enzyme deficient in glycogen storage disease type VI, liver glycogen phosphorylase (PYGL), has been localized by in situ hybridization to 14q21-->q22, near the q21-->q22 band interface. Four additional genes, chromogranin A (CHGA), myosin (MYH7), tRNA proline 2 (TRP2) and c-FOS (FOS) and four random segments, D14S26, D14S12, D14S14 and D14S13 have been more precisely localized. This study also defines a hybrid cell panel with seven mapping intervals, that will be useful for further physical mapping of new loci.

Physical mapping of four serpin genes: alpha 1-antitrypsin, alpha 1-antichymotrypsin, corticosteroid-binding globulin, and protein C inhibitor, within a 280-kb region on chromosome I4q32.1.

Alpha 1-antitrypsin (alpha 1AT; protease inhibitor [PI] locus), alpha 1-antichymotrypsin (alpha 1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3' of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. We have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome l2. The close proximity of these genes has implications for disease-association studies.

Rare deficiency types of alpha 1-antitrypsin: electrophoretic variation and DNA haplotypes.

A deficiency of the plasma protease inhibitor alpha 1-antitrypsin (alpha 1AT), is usually associated with the deficiency allele PI*Z. However, other alleles can also produce a deficiency. Some of these rare deficiency alleles produce a low concentration (3%-15% of normal) of alpha 1AT and include Mmalton, Mduarte, Mheerlen, and Mprocida. Null, or nonproducing, alleles are associated with trace amounts (less than 1%) of plasma alpha 1AT. We have identified, using isoelectric focusing, the deficiency alleles in 222 patients (68 children and 154 adults) with alpha 1AT deficiency. In addition to PI*Z, we found low-producing alleles PI*Mmalton and PI*Mcobalt and four null (PI*QO) alleles. On the basis of a population frequency of .0122 for PI*Z, frequencies for other deficiency alleles are 1.1 x 10(-4) for PI*Mmalton, 2.5 x 10(-5) for PI*Mcobalt (which may be the same as that for PI*Mduarte, and 1.4 x 10(-4) for all null alleles combined. Using 12 polymorphic restriction sites with seven different restriction enzymes, we have obtained DNA haplotypes for each of the rare deficiency types. All of the rare deficiency alleles can be distinguished from PI*Z by their DNA haplotype, and most can be distinguished from each other. DNA haplotypes are useful to indicate the presence of new types of null alleles, to identify genetic compounds for rare deficiency alleles, and to identify the original normal allele from which each deficiency allele is derived.

DNA restriction-site polymorphisms associated with the alpha 1-antitrypsin gene.

Restriction-site variation in and around the alpha 1-antitrypsin gene has been studied using two genomic probes. With use of restriction enzymes SstI, MspI, and AvaII, three polymorphic sites have been described with a 4.6-kb probe in the 5' portion of the gene. With use of a 6.5-kb probe, polymorphisms in the coding and 3' regions of the gene have been detected with AvaII, MaeIII, and TaqI. All of these polymorphisms are of sufficiently high frequency to be useful in genetic mapping studies. The polymorphisms with AvaII and MaeIII (6.5-kb probe) are particularly useful for prenatal diagnosis. PI types and M subtypes tend to be associated with specific DNA haplotypes; there are two different types of DNA haplotypes associated with PI M1. The extent of linkage disequilibrium differs throughout the region of the alpha 1-antitrypsin gene.

Aggregation of plasma Z type alpha 1-antitrypsin suggests basic defect for the deficiency.

The abnormal type of alpha 1-antitrypsin, PI (protease inhibitor) type Z, is associated with inclusion bodies in the liver, which contain non-secreted alpha 1-antitrypsin. Our studies show that Z protein has an inherent tendency to aggregate, even in plasma. Depending upon conditions, from 15 to 70% of the Z protein in plasma was in a high-Mr form, compared with 1.5% of M type alpha 1-antitrypsin. The high-Mr complex in plasma cannot be disaggregated using Triton X detergent or reducing conditions. This increased tendency to aggregate can be explained by the mutation affecting, tertiary structure and salt bridge formation in Z protein. We have observed this same tendency to aggregate for Mmalton alpha 1-antitrypsin, a rarer variant also associated with a plasma deficiency.

Oxidation of plasma alpha 1-antitrypsin in smokers and nonsmokers and by an oxidizing agent.

The inhibitor activity of the protease inhibitor alpha 1-antitrypsin (alpha 1-protease inhibitor) is decreased by oxidizing agents such as those found in cigarette smoke. We have compared specific elastase and specific trypsin inhibitor activity, under defined conditions, in 26 smokers and 26 nonsmokers. Contrary to some previous reports, we have not found a difference between them. The oxidizing agent chloramine T was used to provide an additional oxidant stress for the comparison of plasma from nonsmokers and smokers. Although there was considerable variation between individual subjects in the extent of inactivation of inhibitor activity of alpha 1-antitrypsin, there was no significant difference between nonsmokers and smokers. Apparently there was sufficient antioxidant activity in the plasma of the smokers we tested to prevent the detection of oxidized inactivated alpha 1-antitrypsin.

Functional assessment of genetic variants of alpha 1-antitrypsin.

The specific activity of thirteen genetic variants of the protease inhibitor alpha 1-antitrypsin (alpha 1AT) has been determined. Elastase inhibitor activity was assayed protein substrates (elastin and gelatin) and the synthetic substrate N-tert-butoxy-carbonyl-L-alanine p-nitrophenyl ester. The synthetic substrate alpha-N-benzoyl-DL-arginine p-nitroanilide HCl was used to assay trypsin inhibitor activity. The specific activity of alpha 1AT was expressed as serum inhibition/immunological concentration of alpha 1AT. Sera of PI type FM had reduced specific activity with elastase, but not with trypsin. With the possible exception of MP, no other variants showed significant differences in specific activity when compared with normal PI type M.

Functional assay of alpha 1-antitrypsin in obstructive lung disease.

A decreased concentration of alpha 1-antitrypsin is associated with a high risk for obstructive lung disease. We measured the elastase inhibitory capacity, the most important biologic measure of alpha 1-AT function, using a natural substrate. The gel plate assay that we developed uses a commercial gelatin film and is more sensitive, faster, and cheaper than similar elastin-elastase methods. In serum samples from 76 patients with emphysema, there was a high correlation between the immunologically measured alpha 1-AT and the elastase inhibitory capacity. There was no evidence for a functionally deficient alpha 1-AT in any of these patients.