DNA methylation analysis with respect to prenatal diagnosis of the Angelman and Prader-Willi syndromes and imprinting.

The Angelman (AS) and Prader-Willi syndromes (PWS) are clinically distinct neurobehavioural syndromes resulting from loss of maternal (AS) or paternal contributions (PWS) of imprinted genes within the chromosomal 15q11-q13 region. The molecular diagnosis of both syndromes can be made by a variety of techniques, including DNA methylation, DNA polymorphism and molecular cytogenetic analyses. DNA methylation analysis at three major loci (ZNF127, PW71 and 5' SNRPN) has been successfully used for the postnatal diagnosis of AS and PWS. Methylation analysis, in contrast to other techniques, can reliably be used to diagnose all three major molecular classes (deletion, uniparental disomy and imprinting mutation) of PWS, and three of the four major classes of AS. In this study we demonstrate that methylation analysis can also be successfully used in prenatal diagnosis, by examining specimens obtained from amniocentesis and chorionic villus sampling. Correct prenatal diagnoses were obtained in 24 out of 24 samples using the 5' SNRPN locus; 4 out of 15 using the ZNF127 locus; and 10 out of 18 using the PW71 locus. Therefore, our data indicate that although the DNA methylation imprints of ZNF127 and 5' SNRPN arise in the germline and are present in brain, only 5' SNRPN maintains the imprint in tissues suitable for the prenatal diagnosis of AS and PWS.

Myotonic dystrophy: tissue-specific effect of somatic CTG expansions on allele-specific DMAHP/SIX5 expression.

Myotonic dystrophy (DM), the most common inherited muscle disorder, is caused by a CTG expansion in the 3"-untranslated region of a protein kinase gene ( DMPK ). The complex and variable phenotype is most likely caused by a complex molecular pathogenesis, including deficiency of the DMPK protein, a trans -dominant misregulation of RNA homeostasis and haploinsufficiency of a neighboring homeobox gene [DM locus-associated homeodomain protein (DMAHP )]. Here, we study the allele-specific transcriptional activity of the DMAHP/SIX5 gene in DM patient tissues. Using a quantitative fluorescent RT-PCR assay, we tested allele-specific accumulation of DMAHP/SIX5 transcripts in both total and poly(A)+pools. In muscle biopsies, we found that transcript reductions of DMAHP/SIX5 alleles in cis with CTG expansions correlated with the extent of expansion. A patient with approximately 90 CTG repeats in muscle DNA (normal n < 37) showed a 20% reduction of allele-specific transcript levels, while four other DM patients with larger expansions showed 80% reductions. The effects of the CTG expansions on DMAHP transcription were tissue specific: autopsy tissues from a patient with 1500 repeats showed 80% reductions in muscle and liver; however, RNA from other tissues (lung, aorta, heart conduction tissue, cerebellum) showed 0-20% reductions. Our results suggest that the effect of the CTG repeat on the DMAHP/SIX5 promoter is variable and tissue-specific. Our data are consistent with abnormalities of DMAHP/SIX5 probably having a more prominent role in disease pathogenesis in muscle, liver and brain, but being less important in other tissues.

Neurogenetics in developmental and behavioral pediatrics: advances in molecular diagnosis.

The discovery and characterization of thousands of genes involved in human disorders has the potential for great benefit in patient diagnosis and treatment. A combined approach using molecular genetic testing for some disorders as an adjunct to the clinical evaluation of children with developmental or behavioral features is rapidly occupying a more prominent role in the diagnostic evaluation. In some instances, molecular testing might confirm a genetic etiology in a child initially referred with a DSM-IV diagnosis, but molecular testing might be of limited use in other instances. This review presents advances in the diagnosis of inherited disorders affecting the pediatric population, with an emphasis on those with a developmental/behavioral component. We examine several relatively common disorders in detail (and less common disorders in a more cursory fashion) to elucidate the strengths and weaknesses of molecular diagnosis in clinical practice.

Clinical description of an adult male with psychosis who showed FMR1 gene methylation mosaicism.

Unstable trinucleotide repeat DNA contained in numerous genes has been proposed as the underlying mechanism in the clinical phenomenon of genetic anticipation in fragile X syndrome and other neurodegenerative diseases. No clear evidence has been found for the role of these abnormal trinucleotide repeat expansion-containing genes in schizophrenia or other psychiatric disorders. This report describes an adult male with psychosis who was later found to have methylation mosaicism of the FMR1 gene. We discuss history, examination, and investigation which led to the diagnosis and treatment response of this patient.

Most Jacobsen syndrome deletion breakpoints occur distal to FRA11B.

Recent studies have identified a (CCG)n repeat in the 5' untranslated region of the CBL2 protooncogene (11q23.3) and have demonstrated that expansion of this repeat causes expression of the folate-sensitive fragile site FRA11B. It has also been demonstrated that FRA11B is the site of breakage in some cases of Jacobsen syndrome (JS) involving terminal deletions of chromosome 11q. We report on 2 patients with JS and a 46,XX,del(11)(q23.3) karyotype. In both cases, microsatellite and fluorescence in situ hybridization analyses indicated that the deletion breakpoint was approximately 1.5-3 Mb telomeric to FRA11B. There was no evidence of expansion of the CBL2 (CCG)n repeat in the parents of either patient. The deleted chromosome was of paternal origin in both cases, although it was of maternal origin in the cases reported to be caused by FRA11B. These findings and those in previously reported patients suggest that the breakpoint for most 11q deletions in JS patients is telomeric to FRA11B, which raises the possibility that there may be other fragile sites in 11q23.3 in addition to FRA11B. These findings also support previous evidence that there may be a propensity for breakpoints to differ depending on the parental origin of the deleted chromosome.

Fragile X syndrome and deletions in FMR1: new case and review of the literature.

The fragile X syndrome phenotype of mental retardation is almost always caused by abnormal CGG trinucleotide amplification within the FMR1 gene. Occasionally fragile X syndrome results from point mutations or deletions within or around the FMR1 locus. We have identified a mentally retarded African American male with typical fragile X phenotype and a 300-400 base pair intragenic deletion near the CGG repeat segment, present in his peripheral blood lymphocytes with no apparent mosaicism. His mother, who is not retarded, has a full FMR1 CGG expansion mutation with 700-900 repeats. A review of 23 published cases with FMR1 gene deletions shows full FMR1 mutation in the mother of only 1 other propositus, a male with FMR1 full mutation/premutation/deletion mosaicism of his cultured skin fibroblasts and peripheral blood lymphocytes. The various deletions within FMR1 and their clinical significance are reviewed.

Evaluation of mental retardation: recommendations of a Consensus Conference: American College of Medical Genetics.

A Consensus Conference utilizing available literature and expert opinion sponsored by the American College of Medical Genetics in October 1995 evaluated the rational approach to the individual with mental retardation. Although no uniform protocol replaces individual clinician judgement, the consensus recommendations were as follows: 1. The individual with mental retardation, the family, and medical care providers benefit from a focused clinical and laboratory evaluation aimed at establishing causation and in providing counseling, prognosis, recurrence risks, and guidelines for management. 2. Essential elements of the evaluation include a three-generation pedigree: pre-, peri-, and post-natal history, complete physical examination focused on the presence of minor anomalies, neurologic examination, and assessment of the behavioral phenotype. 3. Selective laboratory testing should, in most patients, include a banded karyotype. Fragile X testing should be strongly considered in both males and females with unexplained mental retardation, especially in the presence of a positive family history, a consistent physical and behavioral phenotype and absence of major structural abnormalities. Metabolic testing should be initialed in the presence of suggestive clinical and physical findings. Neuroimaging should be considered in patients without a known diagnosis especially in the presence of neurologic symptoms, cranial contour abnormalities, microcephaly, or macrocephaly. In most situations MRI is the testing modality of choice. 4. Sequential evaluation of the patient, occasionally over several years, is often necessary for diagnosis, allowing for delineation of the physical and behavioral phenotype, a logical approach to ancillary testing and appropriate prognostic and reproductive counseling.

The elusive Angelman syndrome critical region.

DNA mapping studies in two families provide further information on the Angelman syndrome critical region, which has recently been defined by the gene UBE3A. The first family has probable familial Angelman syndrome with a maternally imprinted inheritance pattern. A 5 year old girl with this disorder has a 14 year old brother and an 11 year old male cousin who have less typical clinical features. DNA microsatellite analysis has shown that the three share a common segment of the same grandpaternal chromosome 15q11-q13 that overlaps with UBE3A. The child with typical Angelman syndrome has an additional maternal recombination 5' to UBE3A. The second family is a mother and son both of whom have mental retardation but no other features of Angelman syndrome despite an extensive DNA deletion on the telomeric side of UBE3A. Together, the two families identify a region between loci D15S210 and D15S986 which forms part of the Angelman syndrome critical region. A new microsatellite (D15S1234) is described which can be used in place of the LS6-1 marker at locus D15S113.

Down syndrome with biparental inheritance of der(14q21q) and maternally derived trisomy 21: confirmation by fluorescent in situ hybridization and microsatellite polymorphism analysis.

Individuals with translocation Down syndrome (DS) often inherit the rearranged chromosome from a carrier parent. DS due to inheritance of one Robertsonian or derivative (14q21q) from one parent and a second der(14q21q) in addition to a free chromosome 21 from the other parent are rarely documented in liveborn infants. Presented here is such a propositus with DS and with a unique karyotype 45,XY,der(14;21) (p11.1;p11.1)pat,der(14;21)(p11.1;q11.1)mat, +21mat. Using conventional chromosome heteromorphisms, fluorescent in situ hybridization (FISH), and microsatellite polymorphism analyses, we established the biparental origin of the 2 der(14q21q) and the maternal origin of the extra chromosome 21 in the patient. A combination of both cytogenetic and molecular genetic techniques also enabled us to show that the 2 der(14q21q) were not identical by descent and hence the parents were nonconsanguineous. It has been a well-established fact that mothers with Robertsonian translocations have higher risk for nondisjunction than do carrier fathers. Our case, wherein the nondisjunctional event occurred in the mother, even though both parents are carriers of a 14;21 Robertsonian translocation, is yet another example of this.

Characterization of the full fragile X syndrome mutation in fetal gametes.

Fragile X syndrome results from the expansion of the CGG repeat in the FMR1 gene. Expansion has been suggested to be a postzygotic event with the germline protected. From an analysis of intact ovaries of full mutation fetuses, we now show that only full expansion alleles can be detected in oocytes (but in the unmethylated state). Similarly, the testes of a 13-week full mutation fetus show no evidence of premutations while a 17-week full mutation fetus exhibits some germ cells with attributes of premutations. These data discount the hypothesis that the germline is protected from full expansion and suggest full mutation contraction in the immature testis. Thus, full expansion may already exist in the maternal oocyte, or postzygotic expansion, if it occurs, arises quite early in development prior to germline segregation.

Carrier testing in the fragile X syndrome: attitudes and opinions of obligate carriers.

This study surveyed obligate carriers of the fragile X syndrome fra(X) to ascertain opinions and attitudes regarding carrier testing. Female carriers of fra(X) syndrome were recruited during their visits to the Fragile X Clinic at Duke University Medical Center. Twenty-eight obligate carriers completed a 48 question structured interview and a visual analog scale (VAS). Strong trends in the responses were identified. Fra(X) syndrome was viewed as a very serious problem and the risk to offspring high. Subjects reported that prior knowledge of carrier status would have changed their reproductive plans. All felt that relatives should be informed about the inheritance of fra(X) syndrome; the mean age given for preferred age to inform their children of the inheritance of fra(X) syndrome was 12 years, and mean age given for optimal timing of carrier testing was 10 years. The women interviewed indicated that growing up with knowledge of their carrier status would have been preferable to learning this information as adults and they endorsed an aggressive approach to informing and testing their children. Further investigation is warranted to determine the psychological consequences of carrier testing for fra(X) syndrome in order to develop appropriate guidelines for testing and informing individuals at risk for fra(X) syndrome.

Instability of the FMR2 trinucleotide repeat region associated with expanded FMR1 alleles.

The fragile sites FRAXA and FRAXE, located approximately 600 kb apart on Xq27.3 and Xq28, respectively, are due to a CGG trinucleotide repeat expansion. Although the expansion mechanism for these and other trinucleotide repeat disorders remains unknown, the similarities between the FRAXA and FRAXE regions suggest a possible association between the 2 sites. DNA from 953 individuals was analyzed to determine the distribution of FRAXE repeat sizes in this population and to ascertain potential association between FRAXA and FRAXE repeat sizes. Thirty-four FMR2 alleles ranging from 3-42 repeats were identified. No FRAXE expansions were found in this population, supporting previous findings that FRAXE expansions are rare. However, in the fragile X syndrome affected group, a FMR2 delection, 2 cases of FRAXE repeat instability and a FRAXE mosaic male were identified. Also, a previously identified, rare FMR2 polymorphism was observed. Statistical analysis showed no correlation between normal FRAXA and FRAXE repeat sizes studied, although there was a significant size difference in larger FMR2 alleles that segregated with expanded FMR1 alleles. These findings support the idea of an association between repeat expansion in the FMR1 gene and instability or deletions in the FMR2 gene.

Inability to induce fragile sites at CTG repeats in congenital myotonic dystrophy.

Myotonic dystrophy (DM) is a trinucleotide repeat syndrome which can contain 50 to over 2,000 CTG repeats in affected individuals, but does not express a fragile site. Although one prior study [Jalal et al., Am J Med Genet 46:441-443, 1993] did not find evidence of fragility at 19q13.3 in six individuals affected with DM using induction protocols for folate sensitive fragile sites, other chemicals may induce fragile site expression at this site. In an attempt to induce fragile sites at 19q13.3, blood cultures from four congenital DM cases and four control individuals treated with fluorodeoxyuridine (folate-sensitive rare fragile sites), bromodeoxyurdine (rare and common fragile sites), aphidicolin (common fragile sites), and 5-azacytidine (common fragile sites) were harvested using routine cytogenetic technique. Slides were solid stained and 100 cells were examined for fragile site expression, particularly on F group chromosomes. The latter were photographed prior to destaining and G-banded to verify chromosome and band location of breakage. No culture conditions induced a fragile site at band 19q13.3 at > 1% expression in patients with congenital DM. Our results suggest that CTG repeats, even when present in > 1,000 copies, may behave differently from other large expansions which are associated with fragile sites. The CTG repeats in DM are not associated with a methylated CpG island, as are folate-sensitive fragile sites, which most likely plays a role in the expression of fragile sites at the trinucleotide repetitive site.

A fragile X male with a broad smear on Southern blot analysis representing 100-500 CGG repeats and no methylation at the EagI site of the FMR-1 gene.

Fragile X DNA studies were carried out on all obligate carriers of a large fragile X family with 10 mentally retarded individuals. One 64-year-old carrier man with an altered FMR-1 allele was not described as being mentally retarded or as having any limitations in function. He was married, raised 8 children, and worked as an auto mechanic. On examination, he had macrocephaly and mild macroorchidism but few of the other typical physical findings of males with fragile X syndrome. His Full Scale IQ is 73, and his Vineland Adaptive Behavior Composite is 73. On the Woodcock-Johnson Psycho-Educational Battery-Revised, he achieved standard scores of 64 in Reading, 55 in Math, and 83 in Knowledge. His DNA findings showed a broad smear on Southern blot analysis of 100-500 CGG repeats and no methylation at the EagI site upstream of the FMR-1 protein coding region. His FMR-1 protein production is 12% of normal. His daughters all have large premutations, with somatic instability in the size of the CGG repeat lengths. They all have evidence of academic underachievement and 2 have physical characteristics frequently described in individuals with fragile X.

Rater reliability of fragile X mutation size estimates: a multilaboratory analysis.

Notwithstanding the use of comparable molecular protocols, description and measurement of the fra(X) (fragile X) mutation may vary according to its appearance as a discrete band, smear, multiple bands, or mosaic. Estimation of mutation size may also differ from one laboratory to another. We report on the description of an mutation size estimate for a large sample of individuals tested for the fra(X) pre- or full mutation. Of 63 DNA samples evaluated, 45 were identified previously as fra(X) pre- or full mutations. DNA from 18 unaffected individuals was used as control. Genomic DNA was extracted from peripheral blood, and DNA fragments from each of four laboratories were sent to a single center where Southern blots were prepared and hybridized with the pE5.1 probe. Photographs from autoradiographs were returned to each site, and raters blind to the identity of the specimens were asked to evaluate them. Raters' estimates of mutation size compared favorably with a reference test. Intrarater reliability was good to excellent. Variability in mutation size estimates was comparable across band types. Variability in estimates was moderate, and was significantly correlated with absolute mutation size and band type.

Longitudinal study of cognitive abilities and adaptive behavior levels in fragile X males: a prospective multicenter analysis.

Retrospective longitudinal studies have noted declines in IQ scores in many but not all fra(X) (fragile X) males and females. We report on a prospective investigation of longitudinal changes in cognitive ability (IQ) and adaptive behavior (DQ) in 24 fra(X) males from four test sites. Individuals who were tested ranged in age from 3-15 years. To determine cognitive ability, all males were administered the Stanford-Binet test (4th Edition). To assess adaptive behavior, all males were evaluated using the Vineland Adaptive Behavior Scales. Mean interest interval was 2.3 years. Using identical DNA protocols, all subjects were identified as bearing the fra(X) mutation. Results showed declines in IQ scores in 18/24 (75%) males. Four males showed no change in scores. Declines in DQ scores were noted in 22/24 (92%) of those tested. DQ scores were higher than IQ scores in 20/24 (83%) subjects. From a descriptive cohort analysis, decreases in IQ scores appear to follow a well-defined, negatively decelerating function. Declines in DQ were steeper and more nearly linear. Declining scores are not indicative of regression of intellectual and/or social skills, but of a relative inability to keep pace with their age-normed cohort. We conclude that the fra(X) mutation affects cognitive abilities in a uniform, nonlinear manner comparable to outcomes observed in earlier retrospective studies. Adaptive behavior also declines, but in a more linear fashion.

Lack of association between mutation size and cognitive/behavior deficits in fragile X males: a brief report.

Previously, researchers reported molecular-neurobehavioral or molecular-cognitive associations in individuals with fra(X) (fragile X) mutation. However, not all investigators have noted molecular-behavioral relationships. Consequently, we examined prospectively 30 fra(X) males age 3-15 years from four testing sites to determine whether there was a relationship between mutation size and degree of either cognitive or adaptive behavior deficit. To measure cognitive abilities, all individuals were administered the Stanford-Binet (4th edition) IQ test. To evaluate adaptive behavior (DQ) skills, all individuals were assessed using the Vineland Adaptive Behavior Scale. To determine fra(X) status, genomic DNA from all individuals was extracted and digested with EcoRI and EagI restriction enzymes. Southern blots were prepared and hybridized with the pE5.1 probe. The Pearson correlation coefficient between full mutation size and composite IQ score revealed a nonsignificant, near-zero association (r = 0.06; P > .76). The Pearson coefficient between mutation size and DQ also showed a nonsignificant, near-zero association (r = 0.06; P > .73). We conclude that while fra(X) mutation produces cognitive and behavior deficits in males who inherit the defective gene, there is no relationship between mutation size and degree of deficit.

Characterization of an unbalanced de novo rearrangement by microsatellite polymorphism typing and by fluorescent in situ hybridization.

Unbalanced de novo rearrangements, difficult to characterize by conventional cytogenetic techniques, may be elucidated by molecular approaches. By dinucleotide repeat polymorphism typing and fluorescence in situ hybridization (FISH), we have defined the composition of an unbalanced de novo translocation (46,XX,15p+) in a child with multiple congenital anomalies. Use of a microsatellite repeat D5S208 (localized to 5p15) and polymerase chain reaction (PCR) analysis confirmed that the extra segment originated from the short arm of chromosome 5. Amplification of the patient's DNA with primers for dinucleotide repeats D5S350 and D5S118 showed that the entire 5p (from 5pter to 5q11) was present in 3 copies. FISH confirmed the trisomic status of 5p, and further revealed the presence of centromeres of both chromosomes 5 and 15 on the rearranged chromosome thus delineating its dicentric nature. This information allowed us to redefine the de novo rearrangement in this patient as 46,XX,dic der(15)t(5;15)(q11;p11).