Inherited Interstitial Duplications of Proximal 15q: Genotype-Phenotype Correlations

We present the cytogenetic, molecular cytogenetic, and molecular genetic results on 20 unrelated patients with an interstitial duplication of the proximal long arm of chromosome 15. Multiple probes showed that the Prader-Willi/Angelman critical region (PWACR) was included in the duplication in 4/20 patients, each ascertained with developmental delay. The duplication was also found in two affected but not in three unaffected sibs of one of these patients. All four probands had inherited their duplication from their mothers, three of whom were also affected. Two of the affected mothers also carried a maternally inherited duplication, whereas the duplication in the unaffected mother and in an unaffected grandmother was paternal in origin, raising the possibility of a parental-origin effect. The PWACR was not duplicated in the remaining 16 patients, of whom 4 were referred with developmental delay. In the 14 families for which parental samples were available, the duplication was inherited with equal frequency from a phenotypically normal parent, mother or father. Comparative genomic hybridization undertaken on two patients suggested that proximal 15q outside the PWACR was the origin of the duplicated material. The use of PWACR probes discriminates between a large group of duplications of no apparent clinical significance and a smaller group, in which a maternally derived PWACR duplication is consistently associated with developmental delay and speech difficulties but not with overt features of either Prader-Willi syndrome or Angelman syndrome.     

Isolation and Chromosomal Localization of a Cornea-Specific Human Keratin 12 Gene and Detection of Four Mutations in Meesmann Corneal Epithelial Dystrophy

Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I keratin.     

To Fire the Train: A Second Malignant-Hyperthermia Gene

During direct sequencing of the first hypervariable segment of the human mitochondrial control region, we identified one individual with a heteroplasmic point mutation at nt 16,256. We used primer extension to analyze the proportions of each mitochondrial haplotype in peripheral blood, buccal cells, and single hair roots from this individual and from eight members of his maternal lineage. Significant levels of heteroplasmy were found in only three individuals, and, in these cases, the proportions of each haplotype were similar in both blood and buccal cells. From the changes in mitochondrial haplotypes within mother-offspring pairs, we calculated that the most likely size of a mitochondrial bottleneck during development was 1-27 segregating units. However, highly variable levels of heteroplasmy were found in single hair roots, even among roots from the same individual. We analyzed a large number of hair roots from one individual and found that the proportion of one haplotype was within a range of 9% to > 99% in different roots. Roots originating from within a small patch of skin had haplotype proportions as variable as those from different areas of skin.     

Mutations of the Fanconi Anemia Group A Gene (FAA) in Italian Patients

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive pancytopenia, congenital malformations, and predisposition to acute myeloid leukemia. At least five complementation groups (FA-A-FA-E) have been identified. The relative prevalence of FA-A has been estimated at an average of approximately 65% but may widely vary according to ethnic background. In Italy, 11 of 12 patients analyzed by cell-fusion studies were assigned to group FA-A, suggesting an unusually high relative prevalence of this FA subtype in patients of Italian ancestry. We have screened the 43 exons of the FAA gene and their flanking intronic sequences in 38 Italian FA patients, using RNA-SSCP. Ten different mutations were detected: three nonsense and one missense substitutions, four putative splice mutations, an insertion, and a duplication. Most of the mutations are expected to cause a premature termination of the FAA protein at various sites throughout the molecule. Four protein variants were also found, three of which were polymorphisms. The missense mutation D1359Y, not found in chromosomes from healthy unrelated individuals, was responsible for a local alteration of hydrophobicity in the FAA protein, and it was likely to be pathogenic. Thus, the mutations so far encountered in the FAA gene are essentially all different. Since screening based on the analysis of single exons by genomic DNA amplification apparently detects only a minority of the mutations, methods designed to detect alterations in the genomic structure of the gene or in the FAA polypeptide may be helpful in the identification of FAA mutations.     

Splicing Defects in the COL3A1 Gene: Marked Preference for 5′ (Donor) Splice-Site Mutations in Patients with Exon-Skipping Mutations and Ehlers-Danlos Syndrome Type IV

Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis.     

Mutation analysis of the RSK2 gene in Coffin-Lowry patients: extensive allelic heterogeneity and a high rate of de novo mutations.

Coffin-Lowry syndrome (CLS) is an X-linked disorder characterized by severe psychomotor retardation, facial and digital dysmorphisms, and progressive skeletal deformations. By using a positional cloning approach, we have recently shown that mutations in the gene coding for the RSK2 serine-threonine protein kinase are responsible for this syndrome. To facilitate mutational analysis, we have now determined the genomic structure of the human RSK2 gene. The open reading frame of the RSK2 coding region is split into 22 exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single-strand conformation polymorphism analysis. We screened 37 patients with clinical features suggestive of CLS. Twenty-five nucleotide changes predicted to be disease-causing mutations were identified, including eight splice-site alterations, seven nonsense mutations, five frameshift mutations, and five missense mutations. Twenty-three of them were novel mutations. Coupled with previously reported mutations, these findings bring the total of different RSK2 mutations to 34. These are distributed throughout the RSK2 gene, with no clustering, and all but two, which have been found in two independent patients, are unique. A very high (68%) rate of de novo mutations was observed. It is noteworthy also that three mutations were found in female probands, with no affected male relatives, ascertained through learning disability and mild but suggestive facial and digital dysmorphisms. No obvious correlation was observed between the position or type of the RSK2 mutations and the severity or particular clinical features of CLS.     

Nucleotide Sequence, Genomic Organization, and Chromosomal Localization of Genes Encoding the Human NMDA Receptor Subunits NR3A and NR3B

The N-methyl-D-aspartate (NMDA) receptors are glutamate-regulated ion channels that are critically involved in important physiological and pathological functions of the mammalian central nervous system. We have identified and characterized the gene encoding the human NMDA receptor subunit NR3A (GRIN3A), as well as the gene (GRIN3B) encoding an entirely novel subunit that we named NR3B, as it is most closely related to NR3A (57.4% identity). GRIN3A localizes to chromosome 9q34, in the region 13-34, and consists of nine coding exons. The deduced protein contains 1115 amino acids and shows 92.7% identity to rat NR3A. GRIN3B localizes to chromosome 19p13.3 and contains, as does the mouse NR3B gene (Grin3b), eight coding exons. The deduced proteins of human and mouse NR3B contain 901 and 900 amino acid residues, respectively (81.6% identity). In situ hybridization shows a widespread distribution of Grin3b mRNA in the brain of the adult rat.     

Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency.

Leigh disease associated with cytochrome c oxidase deficiency (LD[COX-]) is one of the most common disorders of the mitochondrial respiratory chain, in infancy and childhood. No mutations in any of the genes encoding the COX-protein subunits have been identified in LD(COX-) patients. Using complementation assays based on the fusion of LD(COX-) cell lines with several rodent/human rho0 hybrids, we demonstrated that the COX phenotype was rescued by the presence of a normal human chromosome 9. Linkage analysis restricted the disease locus to the subtelomeric region of chromosome 9q, within the 7-cM interval between markers D9S1847 and D9S1826. Candidate genes within this region include SURF-1, the yeast homologue (SHY-1) of which encodes a mitochondrial protein necessary for the maintenance of COX activity and respiration. Sequence analysis of SURF-1 revealed mutations in numerous DNA samples from LD(COX-) patients, indicating that this gene is responsible for the major complementation group in this important mitochondrial disorder.     

The hemochromatosis 845 G-->A and 187 C-->G mutations: prevalence in non-Caucasian populations.

Hemochromatosis, the inherited disorder of iron metabolism, leads, if untreated, to progressive iron overload and premature death. The hemochromatosis gene, HFE, recently has been identified, and characterization of this gene has shown that it contains two mutations that result in amino acid substitutions-cDNA nucleotides 845 G-->A (C282Y) and 187 C-->G (H63D). Although hemochromatosis is common in Caucasians, affecting >=1/300 individuals of northern European origin, it has not been recognized in other populations. The present study used PCR and restriction-enzyme digestion to analyze the frequency of the 845 G-->A and 187 C-->G mutations in HLA-typed samples from non-Caucasian populations, comprising Australian Aboriginal, Chinese, and Pacific Islanders. Results showed that the 845 G-->A mutation was present in these populations (allele frequency 0.32%), and, furthermore, it was always seen in conjunction with HLA haplotypes common in Caucasians, suggesting that 845 G-->A may have been introduced into these populations by Caucasian admixture. 187 C-->G was present at an allele frequency of 2.68% in the two populations analyzed (Australian Aboriginal and Chinese). In the Australian Aboriginal samples, 187 C-->G was found to be associated with HLA haplotypes common in Caucasians, suggesting that it was introduced by recent admixture. In the Chinese samples analyzed, 187 C-->G was present in association with a wide variety of HLA haplotypes, showing this mutation to be widespread and likely to predate the more genetically restricted 845 G-->A mutation.