A simple VNTR-PCR method for detecting maternal cell contamination in prenatal diagnosis.

The effectiveness of variable number tandem repeats (VNTRs) was evaluated in the detection of maternal cell contamination. Nonradioactive PCRs were performed on 30 sets of prenatal tissue using VNTRs as primers. The combination of two VNTRs (YNZ22 and APOB) provided information on all 30 cases, distinguishing maternal-fetal genotype patterns and detecting maternal cell contamination in 5 of 30 prenatal cases. The amplification of these two VNTRs does not require radioactive or fluorescence labeling, and a small gel electrophoresis is sufficient to see the maternal-fetal genotype pattern. By this method, detection of maternal cell contamination in prenatal tissues can be obtained in 1 day, without the use of expensive instruments, thus providing DNA laboratories a very sensitive, rapid, and simple proof pretest on all prenatal tissues before performing the final genetic diagnostic testing.

Asymmetry of methylation with FMR-1 full mutation in two 45,X/46,XX mosaic females associated with normal intellect.

The full FMR-1 mutation is known to cause the fragile X syndrome [Fra(X)], but variable expression in females, including normal to deficient intellect, may be related to random X-inactivation (lyonization). We have evaluated 2 mosaic 45,X/46,XX females who are cytogenetically fra(X) positive, have an FMR-1 full mutation, and are of normal intellect. There were 50% fra(X) chromosomes in the 45,X cells of one of the females; this has not been reported previously. In both patients, there was a strong asymmetry of FMR-1 methylation with the normal allele being totally or 90% unmethylated and the mutant allele being similarly methylated. Thus, the apparent selective inactivation of the full mutant FMR-1 allele appears to have resulted in limited expression with normal intellect. The presence of the fra(X) chromosome in 45,X cells is unique; however, there may be no relationship to the asymmetric inactivation of the mutant allele which could be due to chance or a mechanism yet to be delineated.

Linkage disequilibria among (CA)n polymorphisms in the human dystrophin gene and their implications in carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophies.

Four short tandem repeat loci, characterized by length polymorphisms of (CA)n repeats, have been detected within introns 44, 45, 49, and 50 of the human dystrophin gene. The predicted heterozygosities for these loci range from 72 to 93%, and observed allele numbers range from 6 to 19 in 57 normal chromosomes, revealing their high degree of polymorphism. Evidence for significant disequilibria between the loci within introns 49 and 50 is found. These data appear to be consistent with observations of recombination frequencies between these markers and the length of the intron 44 in relation to the entire region. In addition, these four loci are collectively found to be 100% informative in carrier detection/prenatal diagnosis of Becker and Duchenne muscular dystrophies (B/DMD), whereas scoring the (CA)n markers within introns 45 and 49 alone gives a 99.6% success rate.

Characteristics of intergenerational contractions of the CTG repeat in myotonic dystrophy.

In myotonic dystrophy (DM), the size of a CTG repeat in the DM kinase gene generally increases in successive generations with clinical evidence of anticipation. However, there have also been cases with an intergenerational contraction of the repeat. We examined 1,489 DM parent-offspring pairs, of which 95 (6.4%) showed such contractions in peripheral blood leukocytes (PBL). In 56 of the 95 pairs, clinical data allowed an analysis of their anticipation status. It is surprising that anticipation occurred in 27 (48%) of these 56 pairs, while none clearly showed a later onset of DM in the symptomatic offspring. The contraction occurred in 76 (10%) of 753 paternal transmissions and in 19 (3%) of 736 maternal transmissions. Anticipation was observed more frequently in maternal (85%) than in paternal (37%) transmissions (P < .001). The parental repeat size correlated with the size of intergenerational contraction (r2 = .50, P << .001), and the slope of linear regression was steeper in paternal (-.62) than in maternal (-.30) transmissions (P << .001). Sixteen DM parents had multiple DM offspring with the CTG repeat contractions. This frequency was higher than the frequency expected from the probability of the repeat contractions (6.4%) and the size of DM sib population (1.54 DM offspring per DM parent, in 968 DM parents). We conclude that (1) intergenerational contractions of the CTG repeat in leukocyte DNA frequently accompanies apparent anticipation, especially when DM is maternally transmitted, and (2) the paternal origin of the repeat and the presence of the repeat contraction in a sibling increase the probability of the CTG repeat contraction.

A robotics-assisted procedure for large scale cystic fibrosis mutation analysis.

We describe a convenient, efficient, semiautomated protocol for assaying large numbers of DNA samples for over 20 mutations causing cystic fibrosis. The protocol uses the following: (1) a programmable robotic workstation to perform rapid pipetting and dot-blotting operations, (2) an allele-specific oligonucleotide hybridization in a single water bath without correcting for G+C content of oligonucleotides, and (3) a combinatorial system that allows direct determination of the genotype for more frequent mutations. We have used this system routinely for 16 months for carrier detection and for diagnosis of cystic fibrosis. The method can be readily applied to any combination of allele-specific oligonucleotide assays whether for multiple alleles at one locus or for a few alleles at multiple loci.

Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy.

The myotonic dystrophy mutation has recently been identified; however, the molecular mechanism of the disease is still unknown. The sequence of the myotonin-protein kinase gene was determined, and messenger RNA spliced forms were identified in various tissues. Antisera were developed for analytical studies. Quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to demonstrate that decreased levels of the messenger RNA and protein expression are associated with the adult form of myotonic dystrophy.

Relationship between parental trinucleotide GCT repeat length and severity of myotonic dystrophy in offspring.

OBJECTIVE: To assess the relationship between the GCT repeat number in the myotonic dystrophy gene and the clinical phenotype and examine its predictive utility in prenatal testing. DESIGN: DNA from patients was examined for the length of the myotonic dystrophy GCT repeat region, using both Southern blot analysis and polymerase chain reaction. The results were compared with the clinical onset of disease, as well as with pregnancy outcomes. SETTING: Patient samples were referred to the Kleberg DNA Diagnostic Laboratory at the Baylor College of Medicine for DNA analysis by geneticists and genetic counselors (84%), neurologists (10%), and obstetricians and other specialists (6%). Clinical features including onset of disease and family pedigrees were determined by the referring centers. PATIENTS: A total of 241 patient samples from 118 families referred from primarily genetic or neurological centers for genetic linkage analysis or mutation analysis for myotonic dystrophy. This included 44 families referred for prenatal diagnosis. MAIN OUTCOME MEASURES: A relationship between myotonic dystrophy disease onset and length of the GCT repeat allele, parental origin of the disease allele, and results of prenatal diagnosis predictions of disease status were measured. RESULTS: There is a relationship between increasing repeat length and earlier clinical onset of disease. Essentially all (> 99%) myotonic mutations causing myotonic dystrophy are accounted for by GCT repeat amplification. Congenital myotonic dystrophy occurs with as few as 730 GCT repeats but only with alleles of maternal origin. Maternal GCT repeats were found as low as 75 (asymptomatic) that were amplified to result in a child with congenital myotonic dystrophy. Application of DNA diagnosis to 32 pregnancies provided an accurate method for identification of at-risk fetuses and allele enlargement. CONCLUSIONS: The GCT repeat in myotonic dystrophy is highly mutable. The triplet repeat amplification is highly specific for mutations involving the myotonin protein kinase gene accounting for myotonic dystrophy. The quantitation of triplet repeats can be more sensitive than physical, ophthalmologic, and electromyography examinations since the mutation can be detected in patients without evidence of myotonic dystrophy clinical findings. The length of the triplet expansion is influenced by the sex of the transmitting parent and is related to the clinical onset of disease features. Prenatal measurement of the GCT triplet repeat has utility for families with myotonic dystrophy risk since mutant and normal repeats are distinguishable and the length of mutant repeat alleles is associated with clinical severity. Thus, GCT triplet measurement provides a highly accurate means of detecting the myotonic dystrophy mutation in patients and offers a new reproductive option for families at risk for myotonic dystrophy.

Premature chain termination mutation causing Duchenne muscular dystrophy.

We identified a premature chain termination mutation in two brothers with Duchenne muscular dystrophy and correlated the mutation in one of the brothers with immunologic detection of dystrophin in skeletal muscle. Southern and polymerase chain reaction (PCR) studies of genomic DNA from the affected boys showed no major gene rearrangements. However, the noted absence of a HindIII Southern fragment containing the proximal portion of exon 48 led to the identification of a point mutation that creates a new HindIII restriction site in that exon. Exon 48 was amplified by PCR from DNA of the patients and other family members and digested with HindIII to show the mutation in the two boys and also in their mother and maternal grandmother. Direct DNA sequencing demonstrated a cytosine-to-thymine transition at nucleotide 7163 of dystrophin that converts a glutamine codon (CAA) to an ochre chain termination codon (UAA). This mutation predicts a truncated dystrophin missing the distal spectrin-like repeat region, the cysteine-rich domain, and the carboxy terminal. Immunohistochemistry of skeletal muscle from one of the affected boys revealed membrane-localized dystrophin in the majority of fibers detected by anti-dystrophin antibodies against (1) the amino terminal and (2) part of the spectrin-like repeat region; both regions would be present in the truncated dystrophin predicted by the chain termination mutation. This suggests that the carboxy terminal may not be an absolute requirement for dystrophin membrane localization. Very few muscle fibers also showed peripheral immunostaining using anti-dystrophin antibodies against the carboxy terminal, suggesting gene reversion, suppression, or read-through in these rare fibers.

A transposon-like element in the deletion-prone region of the dystrophin gene.

The central portion of the dystrophin gene locus is a preferential site for deletions causing progressive muscular dystrophy of the Duchenne type (DMD). The nucleotide sequence of a deletion junction fragment from a DMD patient was determined, revealing that the proximal breakpoint of the deletion in intron 43 fell within the sequence of a transposon-like element. This segment, belonging to the THE-1 family of human transposable elements, is normally present in a complete form in intron 43 of the dystrophin gene. The deletion mutation was maternally transmitted and eliminated two-thirds of the THE-1 element. Analysis of DNA from additional DMD patients revealed a second deletion with the proximal breakpoint mapping within the same THE-1 element.

Diagnosis of Duchenne and Becker muscular dystrophies by polymerase chain reaction. A multicenter study.

OBJECTIVE--To assess the efficiency, reliability, and ease of use of DNA diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) using the polymerase chain reaction (PCR). DESIGN--DNA from the patients was screened for deletion mutations using multiplex PCR, and the results were compared with those obtained by Southern blot analysis. The PCR multiplex reaction detects nine specific "hot-spot" exons in the dystrophin gene while the Southern analysis detects 66 specific dystrophin gene restriction fragments. The multiplex reaction requires 50-fold less DNA than Southern analysis and thus is considerably more sensitive. SETTING--Fourteen university-affiliated and private genetic disease diagnostic laboratories. PATIENTS--Male patients with clinical signs of DMD/BMD. Cases were selected for analysis randomly, without knowledge of whether a deletion was present within the dystrophin gene. MAIN OUTCOME MEASURES--The percentage of cases that were detectable by multiplex PCR in comparison with Southern analysis, the frequency, extent, and location of the detected deletion mutations. In some cases, duplication mutations were monitored. RESULTS--The accuracy of a single PCR multiplex amplification (nine exons) was compared with Southern analysis with 10 cDNA probes that cover the full length of the gene. The multiplex PCR analytic method detected 82% of those deletions detected by Southern analysis methods. In one of 745 analyses, the multiplex method suggested a single exon deletion, which was not confirmed by Southern analysis, representing a false-positive rate of 0.013%. CONCLUSIONS--Multiplex PCR represents a sensitive and accurate method for deletion detection of 46% of all cases of DMD/BMD. The method requires 1 day for analysis, is easy to perform, and does not use radioactive tracers. As such, multiplex PCR represents an efficient and rapid method for prenatal or postnatal diagnosis of DMD/BMD.

Triplet repeat mutations in human disease.

Triplet repeats are the sites of mutation in three human heritable disorders, spinal and bulbar muscular atrophy (SBMA), fragile X syndrome, and myotonic dystrophy (DM). These repeats are GC-rich and highly polymorphic in the normal population. Fragile X syndrome and DM are examples of diseases in which premutation alleles cause little or no disease in the individual, but give rise to significantly amplified repeats in affected progeny. This newly identified mechanism of mutation has, so far, been identified in two of the most common heritable disorders, fragile X syndrome and DM, and one rare disease, SBMA.

An unstable triplet repeat in a gene related to myotonic muscular dystrophy.

Synthetic oligonucleotides containing GC-rich triplet sequences were used in a scanning strategy to identify unstable genetic sequences at the myotonic dystrophy (DM) locus. A highly polymorphic GCT repeat was identified and found to be unstable, with an increased number of repeats occurring in DM patients. In the case of severe congenital DM, the paternal triplet allele was inherited unaltered while the maternal, DM-associated allele was unstable. These studies suggest that the mutational mechanism leading to DM is triplet amplification, similar to that occurring in the fragile X syndrome. The triplet repeat sequence is within a gene (to be referred to as myotonin-protein kinase), which has a sequence similar to protein kinases.

Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.

Fragile X syndrome results from mutations in a (CGG)n repeat found in the coding sequence of the FMR-1 gene. Analysis of length variation in this region in normal individuals shows a range of allele sizes varying from a low of 6 to a high of 54 repeats. Premutations showing no phenotypic effect in fragile X families range in size from 52 to over 200 repeats. All alleles with greater than 52 repeats, including those identified in a normal family, are meiotically unstable with a mutation frequency of one, while 75 meioses of alleles of 46 repeats and below have shown no mutation. Premutation alleles are also mitotically unstable as mosaicism is observed. The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox.

Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms.

To improve carrier detection and prenatal diagnosis for Duchenne and Becker muscular dystrophy families, we determined allele frequencies and measures of variation for four (dC-dA)n.(dG-dT)n loci identified within a deletion-prone region of the human dystrophin gene. The loci are highly polymorphic, with predicted heterozygosities of 71.6%-93.3%. Direct DNA sequence analysis of the (dC-dA)n.(dG-dT)n locus in intron 49 revealed an additional length polymorphism which varies by single-basepair increments, is adjacent to the dinucleotide repeat block, and enhances the polymorphic content of this marker. The four (dC-dA)n.(dG-dT)n loci are each easily amplified by PCR in two diplex reactions. The variability of allele lengths at these loci makes them ideal for carrier detection and prenatal diagnosis, often providing diagnostic information when RFLP analysis is uninformative. These markers have aided in identification of deletion mutations, exclusion of maternal cell contamination of chorionic villus samples, confirmation of paternity, and mapping of gene recombinations. The allele identification of these loci can be performed either with a radiolabel or with an automated, nonradioactive, fluorescent gel detection system.

9p monosomy in a patient with Gilles de la Tourette's syndrome.

Gilles de la Tourette's syndrome (GTS) is a genetic disorder characterized by multiple motor and vocal tics, obsessive-compulsive disorder, and attention-deficit disorder. Family studies support the presence of an autosomal dominant gene; however, to date, an assignment for the GTS locus has not been made. We present the case of a boy with GTS and a deletion of the terminal portion of the short arm of chromosome 9, del(9)(qter----p2304:).

Discordance of muscular dystrophy in monozygotic female twins: evidence supporting asymmetric splitting of the inner cell mass in a manifesting carrier of Duchenne dystrophy.

In 1990, Richards et al. reported dramatically skewed lyonization in a set of female monozygotic twins heterozygous for Duchenne muscular dystrophy (DMD). The skewed inactivation pattern was symmetrical in opposite directions, one twin being affected with DMD, the other one being normal. Here, we report an additional set of female monozygotic twins heterozygous for a mutation at the dystrophin locus. Similarly, one shows a manifesting carrier phenotype while one is normal. However, unlike the previous report, we find a skewed X inactivation pattern only in the affected twin, while the normal twin showed a random X inactivation pattern. Our results lend considerable experimental support for the models of twinning and X inactivation recently outlined by Nance in 1990, in that these twins probably represent asymmetric splitting of the inner cell mass (ICM): The affected twin likely arose when a small proportion of the ICM split off after lyonization had occurred. In this situation, the original ICM could give rise to the normal twin with random lyonization, while the newly split cells would experience catch-up growth and lead to the affected twin. Genetic studies of this family showed that the specific dystrophin gene mutation was an exon duplication that arose sporadically in the paternally derived X chromosome.

Fabry disease in a large Nova Scotia kindred: carrier detection using leucocyte alpha-galactosidase activity and an NcoI polymorphism detected by an alpha-galactosidase cDNA clone.

Fabry disease is an X linked recessive disorder of glycosphingolipid metabolism resulting from a deficiency of the lysosomal hydrolase alpha-galactosidase (alpha-gal). Measurement of the enzyme activity, however, is not an accurate method for identification of female carriers among at risk relatives of affected males. The alpha-gal cDNA and gene have been cloned previously and found to provide useful probes for the molecular analysis of affected families but these clones have not been available to us. Thus, to analyse Fabry disease in Nova Scotia, especially within a large kindred known to contain 30 affected males and 50 possible carrier females, we isolated an independent cDNA for alpha-gal. Using this clone as a probe, the mutation in the Nova Scotia kindred was shown not to be a major DNA alteration, but was found to be linked to the rarer allele (frequency 0.20) of the polymorphic NcoI site located 3' to the gene. Affected males from two Nova Scotia families who cannot be associated with the kindred by history were also found to have the rarer NcoI allele, which suggests they are, in fact, part of the kindred. The coupling of the mutation to an infrequent marker also helped carrier identification in the kindred where all of 17 obligate carriers examined, including six who were not identified as carriers by enzyme assays, were found to be heterozygous for the RFLP. Thus, DNA analysis can be used for presymptomatic and prenatal diagnosis in most portions of the Nova Scotia kindred affected with Fabry disease.

The G1 interval in the mammalian cell cycle: dual control by mass accumulation and stage-specific activities.

The temporal determinants of the G1 cell cycle interval were investigated using nine mammalian cell lines. In each case, cells were allowed to proliferate for many cell cycles under conditions that slowed progress through S phase without an equivalent impairment of overall mass accumulation. This disproportionate inhibition of progress through the cell cycle caused newly produced cells to be more massive than usual. Under these growth conditions, the determinants of the length of the G1 interval became evident. For two cell lines, HeLa S3 and NIH 3T3, a protracted S phase, and the resultant increase in mass, resulted in a dramatically shortened G1 interval. Thus, for these cell lines, a major portion of G1 time exists to accommodate mass accumulation needed to initiate the subsequent S phase. Nevertheless, under conditions that protracted S phase and shortened the G1 interval, cells still exhibited a measurable G1 time, reflecting the stage-specific activities within G1. One activity that may be responsible for this obligatory G1 time is the synthesis of a labile protein. For other cells studied here, protraction of S phase also caused proliferating cells to become more massive, but in these cases there was no diminution of the G1 time. For these cells, the entire G1 interval must accommodate G1-specific activities necessary to initiate a new cell cycle. A unifying view of the G1 interval recognizes the two distinct influences that determine the time spent in G1: the need to accumulate sufficient mass to initiate a new DNA-division sequence; and the stage-specific events necessary for the subsequent S phase. The length of the G1 interval is dictated by the longer of these two time-consuming activities.

Improved molecular diagnostics for ornithine transcarbamylase deficiency.

Since the cloning of the cDNA for X-linked ornithine transcarbamylase (OTC) in 1984, diagnostic accuracy of OTC deficiency for prenatal and carrier detection has been greatly improved by the use of linkage analysis. However, the use of RFLP-based diagnosis is limited in this and in other new mutation diseases. Here we report both the use of direct mutation detection by new PCR-based techniques and our experience with linkage-based diagnosis in 18 families. We have previously reported the use of chemical mismatch cleavage to detect mutations first in amplified mRNA and then in genomic DNA of patients. This technique has now been utilized for prenatal diagnosis. Primers for specific amplification of OTC exons 1, 3, 5, 9, and 10 have been developed and been employed to map deletions of the OTC gene in two families. These primers also have been used to detect alterations in the TaqI sites found in exons 1, 3, 5, and 9. Four novel mutations of the OTC gene leading to abolition of a TaqI site in the OTC cDNA were discovered. One of these mutations is in exon 1; two lie in exon 3; and one is in exon 9. In addition, we have used the PCR products as probes to identify the exon-specific bands seen on Southern blots and to map the polymorphic BamHI and MspI sites, which are commonly used for linkage analysis. This information will facilitate the interpretation of altered band patterns seen in deletion cases and in cases of point mutations affecting restriction sites. Utilization of the appropriate combination of these molecular techniques permitted accurate diagnostic evaluations in 17 of 18 families.