Intron/exon structure confirms that mouse Zfy1 and Zfy2 are members of the ZFY gene family.

Zfy1 and Zfy2 are homologous zinc finger genes on the mouse Y Chromosome. To ask whether these genes are properly classified as members of the ZFY family, we have characterized and compared their genomic organization to that of mouse Zfx, human ZFX, and human ZFY. We show that Zfy1 has 11 exons distributed across at least 56 kb, and Zfy2 has a minimum of 9 exons distributed across at least 52 kb. The Zfy2 locus contains regions similar in size and sequence to all 11 exons of Zfy1, plus an additional 5' UTR exon. All splice sites conform to the GT-AG rule. There are two instances of additional AG dinucleotides immediately 5' of 3' splice sites. Zfy1 and Zfy2 are homologous to other ZFY family members within the coding region, but the untranslated regions show no sequence similarity. Within the coding region, there is conservation of exon length and splice sites, with each splice preceding the second nucleotide of a codon. We conclude that Zfy1 and Zfy2 are indeed members of the ZFY family, which has evolved from a single common ancestral gene.

Severe factor VII deficiency due to a mutation disrupting a hepatocyte nuclear factor 4 binding site in the factor VII promoter.

Although small deletions, splice site abnormalities, missense, and nonsense mutations have been identified in patients with factor VII deficiency, there have been no reports of mutations in the factor VII promoter. We investigated a girl with factor VII levels that were less than 1% of normal in association with a severe bleeding diathesis. The patient is homozygous for a T to G transversion that occurs 61 bp before the translation start site. This nucleotide is in a sequence that is an hepatocyte nuclear factor 4 (HNF-4) binding site within the factor VII promoter (ACTTTG AE-->ACGTTG). Using gel mobility shift assays, we show that the mutation disrupts the binding of HNF-4 to its cognate binding site. In growth hormone reporter gene assays, the activity of a plasmid containing the mutant promoter was 6.7% of the wild-type promoter plasmid. Although HNF-4 was able to transactivate the wild-type factor VII promoter 5.4-fold in HeLa cells, no transactivation could be shown with the mutant promoter. These findings indicate that HNF-4 exerts a major positive regulatory effect on factor VII expression and provides in vivo evidence that binding of this transcription factor is critical for normal factor VII expression.

Frequencies of cystic fibrosis mutations in the Maine population: high proportion of unknown alleles in individuals of French-Canadian ancestry.

Cystic fibrosis (CF) is one of the most common severe autosomal recessive disorders in Caucasian populations. A mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene causes this disorder. Reported here is the first analysis of CF mutations in the Maine population. We have screened 263 CF chromosomes for 16 previously reported mutations. Analysis of DNA from 124 apparently unrelated CF patients and 15 obligate carrier parents (whose partner and affected child were unavailable for study) resulted in the identification of 91% of the CF alleles and complete genotyping of 85% of the patients. The frequencies (%) of these mutations in the Maine population are delta F508 (75% of the chromosomes), G85E (0.76), R117H (0.76), I148T (1.1), 621 + 1G --> T (1.1), 711 + 1G --> T (3.0), A455E (1.1), 1717-1G --> A (1.1), G542X (1.9), G551D (1.9), R560T (0.76), Y1092X (0.38), W1282X (0.38), and N1303K (1.5). The exon 10 mutation, delta I507, and the exon 11 mutation, R553X, were not observed. Surprisingly, whereas only 5% of the alleles remain unidentified in the non-French population, the unidentified proportion in the French population is 19%. CF testing for the Maine population will be further improved as the as yet unidentified CF mutations in this population are characterized.

A combined cytogenetic and molecular approach for diagnosing delayed puberty.

A phenotypic female aged 15 4/12 years was referred because of delayed puberty and short stature. Chromosomal analysis of peripheral blood leukocytes revealed two subpopulations of cells. The modal cell line was hypodiploid with a missing X chromosome while the other cell line was diploid with one X chromosome and a G-sized chromosome that resembled a Y chromosome in morphology. Subsequent fluorescent in situ hybridization yielded results consistent with the above conventional cytogenetic studies. To provide unequivocal evidence of the Y-chromosome material, molecular analyses using the polymerase chain reaction and various primers were carried out which identified an intact short arm and centromere of the Y chromosome and structurally altered long arms. A laparoscopic bilateral gonadectomy, performed because of the risk of neoplasia, also yielded cells with both 45,X and 46,XY karyotypes. The present report thus illustrates the usefulness of a combined approach for diagnosing delayed puberty.