CYP2B6 metabolizes many drugs, and its expression varies greatly. CYP2B6 genotype-phenotype associations were determined using human livers that were biochemically phenotyped for CYP2B6 (mRNA, protein, and CYP2B6 activity), and genotyped for CYP2B6 coding and 5Ј-flanking regions. CYP2B6 expression differed significantly between sexes. Females had higher amounts of CYP2B6 mRNA (3.9-fold, P Ͻ 0.001), protein (1.7-fold, P Ͻ 0.009), and activity (1.6-fold, P Ͻ 0.05) than did male subjects. Furthermore, 7.1% of females and 20% of males were poor CYP2B6 metabolizers. Striking differences among different ethnic groups were observed: CYP2B6 activity was 3.6-and 5.0-fold higher in Hispanic females than in Caucasian (P Ͻ 0.022) or African-American females (P Ͻ 0.038). Ten single nucleotide polymorphisms (SNPs) in the CYP2B6 promoter and seven in the coding region were found, including a newly identified 13072AϾG substitution that resulted in an Lys139Glu change. Many CYP2B6 splice variants (SV) were observed, and the most common variant lacked exons 4 to 6. A nonsynonymous SNP in exon 4 (15631GϾT), which disrupted an exonic splicing enhancer, and a SNP 15582CϾT in an intron-3 branch site were correlated with this SV. The extent to which CYP2B6 variation was a predictor of CYP2B6 activity varied according to sex and ethnicity. The 1459CϾT SNP, which resulted in the Arg487Cys substitution, was associated with the lowest level of CYP2B6 activity in livers of females. The intron-3 15582CϾT SNP (in significant linkage disequilibrium with a SNP in a putative hepatic nuclear factor 4 (HNF4) binding site) was correlated with lower CYP2B6 expression in females. In conclusion, we found several common SNPs that are associated with polymorphic CYP2B6 expression.

Microdeletions of a region termed the "imprinting center" (IC) in chromosome 15q11-q13 have been identified in several families with Prader-Willi syndrome (PWS) or Angelman syndrome who show epigenetic inheritance for this region that is consistent with a mutation in the imprinting process. The IC controls resetting of parental imprints in 15q11-q13 during gametogenesis. We have identified a larger series of cases of familial PWS, including one case with a deletion of only 7.5 kb, that narrows the PWS critical region to <4. 3 kb spanning the SNRPN gene CpG island and exon 1. Identification of a strong DNase I hypersensitive site, specific for the paternal allele, and six evolutionarily conserved (human-mouse) sequences that are potential transcription-factor binding sites is consistent with this region defining the SNRPN gene promoter. These findings suggest that promoter elements at SNRPN play a key role in the initiation of imprint switching during spermatogenesis. We also identified three patients with sporadic PWS who have an imprinting mutation (IM) and no detectable mutation in the IC. An inherited 15q11-q13 mutation or a trans-factor gene mutation are unlikely; thus, the disease in these patients may arise from a developmental or stochastic failure to switch the maternal-to-paternal imprint during parental spermatogenesis. These studies allow a better understanding of a novel mechanism of human disease, since the epigenetic effect of an IM in the parental germ line determines the phenotypic effect in the patient.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurobehavioral disorders that most often arise from a 4-Mb deletion of chromosome 15q11-q13 during paternal or maternal gametogenesis, respectively. At a de novo frequency of approximately.67-1/10,000 births, these deletions represent a common structural chromosome change in the human genome. To elucidate the mechanism underlying these events, we characterized the regions that contain two proximal breakpoint clusters and a distal cluster. Novel DNA sequences potentially associated with the breakpoints were positionally cloned from YACs within or near these regions. Analyses of rodent-human somatic-cell hybrids, YAC contigs, and FISH of normal or rearranged chromosomes 15 identified duplicated sequences (the END repeats) at or near the breakpoints. The END-repeat units are derived from large genomic duplications of a novel gene (HERC2), many copies of which are transcriptionally active in germline tissues. One of five PWS/AS patients analyzed to date has an identifiable, rearranged HERC2 transcript derived from the deletion event. We postulate that the END repeats flanking 15q11-q13 mediate homologous recombination resulting in deletion. Furthermore, we propose that active transcription of these repeats in male and female germ cells may facilitate the homologous recombination process.

Patients with disorders involving imprinted genes such as Angelman syndrome (AS) and Prader-Willi syndrome (PWS) can have a mutation in the imprinting mechanism. Previously, we identified an imprinting center (IC) within chromosome 15q11-ql3 and proposed that IC mutations block resetting of the imprint, fixing on that chromosome the parental imprint (epigenotype) on which the mutation arose. We now describe four new microdeletions of the IC, the smallest (6 kb) of which currently defines the minimal region sufficient to confer an AS imprinting mutation. The AS deletions all overlap this minimal region, centromeric to the PWS microdeletions, which include the first exon of the SNRPN gene. None of five genes or transcripts in the 1.0 Mb vicinity of the IC (ZNF127, SNRPN, PAR-5, IPW, and PAR-1), each normally expressed only from the paternal allele, was expressed in cells from PWS imprinting mutation patients. In contrast, AS imprinting mutation patients show biparental expression of SNRPN and IPW but must lack expression of the putative AS gene 250-1000 kb distal of the IC. These data strongly support a model in which the paternal chromosome of these PWS patients carries an ancestral maternal epigenotype, and the maternal chromosome of these AS patients carries an ancestral paternal epigenotype. The IC therefore functions to reset the maternal and paternal imprints throughout a 2-Mb imprinted domain within human chromosome 15q11-q13 during gametogenesis.