Peter K. Rogan

Chromosome Breakage in the Prader-Willi and Angelman Syndromes Involves Recombination between Large, Transcribed Repeats at Proximal and Distal Breakpoints

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.

The frequency of uniparental disomy in Prader-Willi syndrome: Implications for molecular diagnosis

BACKGROUND Prader-Willi syndrome is a genetic disorder characterized by infantile hypotonia, obesity, hypogonadism, and mental retardation, but it is difficult to diagnose clinically in infants and young children. In about two thirds of patients, a cytogenetically visible deletion can be detected in the paternally derived chromosome 15 (15q11q13). Recently, patients with Prader-Willi syndrome have been described who do not have the cytogenetic deletion but instead have two copies of the 15q11q13 region that are inherited from the mother (with none inherited from the father). This unusual form of inheritance is known as maternal uniparental disomy. Using molecular genetic techniques, we sought to determine the frequency of uniparental disomy in Prader-Willi syndrome. METHODS We performed molecular analyses using DNA markers within 15q11q13 and elsewhere on chromosome 15 in 30 patients with Prader-Willi syndrome who had no cytogenetically visible deletion. We also studied their parents. Three patients with Prader-Willi syndrome who had a cytogenetic deletion served as controls. RESULTS In 18 of the 30 patients without a cytogenetic deletion (60 percent), we demonstrated the presence of maternal uniparental disomy for chromosome 15 and its association with advanced maternal age. In another eight patients (27 percent), we identified large molecular deletions. The remaining four patients (13 percent) had evidence of normal biparental inheritance for chromosome 15; three of these patients were the only ones in the study who had some atypical clinical features. CONCLUSIONS In about 20 percent of all cases, Prader-Willi syndrome results from the inheritance of both copies of chromosome 15 from the mother (maternal uniparental disomy). With the combined use of cytogenetic and molecular techniques, the genetic basis of Prader-Willi syndrome can be identified in up to 95 percent of patients.

Information Analysis of Human Splice Site Mutations

Splice site nucleotide substitutions can be analyzed by comparing the individual information contents (Ri, bits) of the normal and variant splice junction sequences [Rogan and Schneider, 1995]. In the present study, we related splicing abnormalities to changes in Ri values of 111 previously reported splice site substitutions in 41 different genes. Mutant donor and acceptor sites have significantly less information than their normal counterparts. With one possible exception, primary mutant sites with <2.4 bits were not spliced. Sites with Ri values ≥2.4 bits but less than the corresponding natural site usually decreased, but did not abolish splicing. Substitutions that produced small changes in Ri probably do not impair splicing and are often polymorphisms. The Ri values of activated cryptic sites were generally comparable to or greater than those of the corresponding natural splice sites. Information analysis revealed preexisting cryptic splice junctions that are used instead of the mutated natural site. Other cryptic sites were created or strengthened by sequence changes that simultaneously altered the natural site. Comparison between normal and mutant splice site Ri values distinguishes substitutions that impair splicing from those which do not, distinguishes null alleles from those that are partially functional, and detects activated cryptic splice sites. Hum Mutat 12:153–171, 1998.

Nondisjunction of human acrocentric chromosomes : studies of 432 trisomic fetuses and liveborns

The present report summarizes molecular studies on the parent and meiotic stage of origin of the additional chromosome in 432 fetuses or liveborns with an additional chromosome 13, 14, 15, 21, or 22. Our studies suggest that there is little variation in the origin of nondisjunction among the five acrocentric trisomies and that there is no association between the origin of nondisjunction and the likelihood of survival to term of the trisomic conceptus. The proportion of cases of paternal origin was similar among the five trisomies: 12% for trisomy 13, 17% for trisomy 14, 12% for trisomy 15, 9% for trisomy 21, and 11% for trisomy 22. The stage of nondisjunction was also similar among the five trisomies, with the majority of cases of maternal origin being due to nondisjunction at meiosis I, whereas for paternally derived cases, nondisjuction occurred primarily at meiosis II.

Clinical spectrum and molecular diagnosis of Angelman and Prader-Willi syndrome patients with an imprinting mutation

Recent studies have identified a new class of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients who have biparental inheritance, but neither the typical deletion nor uniparental disomy (UPD) or translocation. However, these patients have uniparental DNA methylation throughout 15q11-q13, and thus appear to have a mutation in the imprinting process for this region. Here we describe detailed clinical findings of five AS imprinting mutation patients (three families) and two PWS imprinting mutation patients (one new family). All these patients have essentially the classical clinical phenotype for the respective syndrome, except that the incidence of microcephaly is lower in imprinting mutation AS patients than in deletion AS patients. Furthermore, imprinting mutation AS and PWS patients do not typically have hypopigmentation, which is commonly found in patients with the usual large deletion. Molecular diagnosis of these cases is initially achieved by DNA methylation analyses of the DN34/ZNF127, PW71 (D15S63), and SNRPN loci. The latter two probes have clear advantages in the simple molecular diagnostic analysis of PWS and AS patients with an imprinting mutation, as has been found for typical deletion or UPD PWS and AS cases. With the recent finding of inherited microdeletions in PWS and AS imprinting mutation families, our studies define a new class of these two syndromes. The clinical and molecular identification of these PWS and AS patients has important genetic counseling consequences.

Maternal uniparental disomy of chromosome 14 confined to an interstitial segment (14q23-14q24.2)

Maternal uniparental disomy for the complete long arm of chromosome 14 has been reported in 14 patients to date and is associated with a specific pattern of malformation. We report a child with clinical features of this syndrome who exhibits maternal uniparental disomy confined to a specific interstitial segment of chromosome 14.

Daytime sleepiness and REM abnormalities in Prader-Willi syndrome: evidence of generalized hypoarousal.

The aim of this study was to clarify the nature of the sleep abnormalities (excessive daytime sleepiness [EDS] and rapid eye movement [REM] sleep alterations) in Prader-Willi; Syndrome (PWS). Eight PWS patients, 15 normal, 16 narcoleptic, and 16 obese subjects were recorded in the sleep laboratory, both during daytime and nighttime. A principal-finding was that EDS in PWS was associated with an increased amount and depth of sleep. In PWS patients with EDS, compared to those PWS patients without EDS or the narcoleptic, obese, and normal groups, there were significant decreases in wakefulness and increases in percentage of sleep time (ST) and slow-wave sleep (SWS) both during daytime and nighttime testing. Also, in the adult PWS subjects (n = 6), in contrast to normal narcoleptic subjects, intensity of EDS was correlated with increased nocturnal percentage of ST and SWS and % SWS was positively correlated with % ST (both during daytime and nighttime testing). Another principal finding was that in PWS there is a unique alteration of the distribution of REM sleep in relation to controls. PWS patients with EDS or shortened nocturnal REM latencies showed a significantly increased number of REM periods, and a decreased average REM interval between REM periods compared to PWS patients with nonshortened nocturnal REM latencies or to the three control groups. Our data suggest that EDS and REM abnormalities in PWS are not manifestations of a narcoleptic-type syndrome or consequences of obesity. We propose that generalized 24-hour hypoarousal is the primary mechanism underlying the sleep abnormalities in PWS patients.

Exon Skipping in IVD RNA Processing in Isovaleric Acidemia Caused by Point Mutations in the Coding Region of the IVD Gene

Isovaleric acidemia (IVA) is a recessive disorder caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). We have reported elsewhere nine point mutations in the IVD gene in fibroblasts of patients with IVA, which lead to abnormalities in IVD protein processing and activity. In this report, we describe eight IVD gene mutations identified in seven IVA patients that result in abnormal splicing of IVD RNA. Four mutations in the coding region lead to aberrantly spliced mRNA species in patient fibroblasts. Three of these are amino acid altering point mutations, whereas one is a single-base insertion that leads to a shift in the reading frame of the mRNA. Two of the coding mutations strengthen pre-existing cryptic splice acceptors adjacent to the natural splice junctions and apparently interfere with exon recognition, resulting in exon skipping. This mechanism for missplicing has not been reported elsewhere. Four other mutations alter either the conserved gt or ag dinucleotide splice sites in the IVD gene. Exon skipping and cryptic splicing were confirmed by transfection of these mutations into a Cos-7 cell line model splicing system. Several of the mutations were predicted by individual information analysis to inactivate or significantly weaken adjacent donor or acceptor sites. The high frequency of splicing mutations identified in these patients is unusual, as is the finding of missplicing associated with missense mutations in exons. These results may lead to a better understanding of the phenotypic complexity of IVA, as well as provide insight into those factors important in defining intron/exon boundaries in vivo.

Racial differences in allelic distribution at the human pulmonary surfactant protein B gene locus (SP-B).

Variable numbers of composite repetitive motifs are found in different individuals within intron 4 of the surfactant protein B (SP-B) gene (Biochem J. 1995;305:583). This study tests the hypothesis that the distribution of SP-B alleles differs among racial/ethnic groups. A total of 412 SP-B alleles were analyzed: 206 from Caucasian, 68 from African-American, and 138 from Nigerian individuals. Twelve groups of alleles (A-L) carrying 3 to 18 motifs were found. The distribution of the 12 alleles in the Caucasian group differs from that found in the Nigerian (p < .001) and African-American (p < .001) populations. The overall distribution of alleles between the African-American and the Nigerian populations were not statistically different. Specific alleles were also present in different proportions among the groups studied. For example, the most common allele (allele E) in all three populations is present at a significantly higher frequency in Caucasians than in the other two populations, but its frequency does not differ from the Nigerian and African-American groups. A less frequent allele, H, also differs significantly when Caucasians are compared with each of the other two populations, but the frequency of this allele is comparable between the African-American and Nigerian populations. To assess the importance of having comparable racial composition between the control and the case groups, a group of African-Americans with respiratory distress syndrome (RDS) (n = 40) was compared with the African American and the Caucasian groups studied above. No significant difference was observed between the racially matched groups but a significant difference (p = .006) was observed between the racially mixed groups. The results indicate that the distribution of SP-B alleles differs between the racial groups but not between the ethnic groups studied. Thus, racial composition of the groups under study is important when considering whether particular alleles at this locus predispose to inherited disorders.

Relationship of sleep abnormalities to patient genotypes in Prader-Willi syndrome

To assess whether sleep abnormalities are related to the genetic abnormalities in Prader-Willi Syndrome (PWS), we performed polysomnographic studies (nighttime and daytime) and determined the chromosome 15 genotypes in eight patients with PWS. Four patients demonstrated sleep onset REM periods (SOREM), and five met the objective polysomnographic criteria for severe or moderate excessive daytime sleepiness (EDS). Three of the four patients with SOREM displayed a paternally derived deletion of chromosome 15q11-q13, whereas the fourth exhibited maternal uniparental heterodisomy in this chromosomal region (UPD). Two of the four patients that did not display SOREM carried paternally derived deletions; the remaining two demonstrated UPD. Four of the five patients with EDS displayed paternal deletions, and the fifth exhibited UPD. One of three patients without evidence of EDS demonstrated paternal deletion; the remaining two showed UPD. Although neither EDS nor SOREM was not consistently associated with a specific genetic abnormality, these phenotypes may be more common in patients with paternal deletions than in those UPD. Sleep abnormalities in PWS cannot be explained by a single genetic model.