Impact of TP53 mutation variant allele frequency on phenotype and outcomes in myelodysplastic syndromes.

Although next-generation sequencing has allowed for the detection of somatic mutations in myelodysplastic syndromes (MDS), the clinical relevance of variant allele frequency (VAF) for the majority of mutations is unknown. We profiled TP53 and 20 additional genes in our training set of 219 patients with MDS or secondary acute myeloid leukemia with findings confirmed in a validation cohort. When parsed by VAF, TP53 VAF predicted for complex cytogenetics in both the training (P=0.001) and validation set (P<0.0001). MDS patients with a TP53 VAF > 40% had a median overall survival (OS) of 124 days versus an OS that was not reached in patients with VAF <20% (hazard ratio (HR), 3.52; P=0.01) with validation in an independent cohort (HR, 4.94, P=0.01). TP53 VAF further stratified distinct prognostic groups independent of clinical prognostic scoring systems (P=0.0005). In multivariate analysis, only a TP53 VAF >40% was an independent covariate (HR, 1.61; P<0.0001). In addition, SRSF2 VAF predicted for monocytosis (P=0.003), RUNX1 VAF with thrombocytopenia (P=0.01) and SF3B1 with ringed sideroblasts (P=0.001). Together, our study indicates that VAF should be incorporated in patient management and risk stratification in MDS.

Microsatellites: perspectives and potentials of mass spectrometric analysis.

Mass spectrometry is a powerful analytical tool in biotechnology. The 'soft' ionization and desorption technologies matrix-assisted laser desorption/ionization and electrospray ionization have enabled mass spectrometric analysis of large biomolecules, such as proteins and nucleic acid amplification products, and paved the way for mass spectrometry to become a leading technology in current genomics and proteomics efforts. Large-scale analysis of single nucleotide polymorphisms by mass spectrometry has been commercially established. This article reviews applications of mass spectrometry for microsatellite analysis. Features and capabilities of the two most prominent techniques, matrix assisted laser desorption/ionization and electrospray-ionization mass spectrometry, are compared and their potential to address the limitations of conventional microsatellite analysis based on comparison of gel electrophoretic mobilities is explored.

Diploid/triploid mosaic placenta with fetus. Towards A better understanding of 'partial moles'.

We describe a case of partial molar change in a placenta that was associated with a normal female fetus who died in utero. The analysis of molar and normal placental tissue, as well as the karyotypic study of amnionic fluid indicate a complex origin of this conceptus. We review the possible mechanisms leading to this pregnancy and the general topic of partial hydatidiform mole. The formation of moles is complex and it is not easily divisible into so-called partial and complete hydatidiform moles. Rather, individual genetic study is needed to make an accurate diagnosis because macroscopic or microscopic examination alone fails to assess the complexity of these entities.

Partial molar transformation of the placenta of presumably monozygotic twins.

A pregnancy with one normal female fetus and a placenta that was divided into halves, one normal the other molar, is described. Genetic analysis shows the molar component to be hyperdiploid/tetraploid but having an identical DNA composition as the normal twin. Because there was no trophoblastic proliferation and the hyperdiploid cells were confined to the villous stroma, and because the molar component was still being perfused by diploid vessels from the normal twin, we believe the mole is derived from polyploidization of the mesenchymal epiblast in a monozygotic twin pregnancy.

Ring chromosome X in a child with manifestations of Kabuki syndrome.

A female patient with the karyotype 45,X/46, X, r(X)(p11.2 q13) and severe developmental delay, prominent fingertip pads, long palpebral fissures, short stature, and history of hypotonia had a phenotype reminiscent of Kabuki syndrome. We hypothesized that overexpression of X chromosome-derived sequences might be associated with the Kabuki-like phenotype observed. The nature and parental origin of this small-ring X were ascertained using a combination of genotyping with microsatellite markers and quantitative Southern blotting. PCR-based genotyping demonstrated heterozygosity at X-linked loci SBMA (Xq11-q12) and DXS227 (Xq13.1). Hemizygosity was observed at several loci: DMD STR-49 (Xp21.2), DXS1003 (Xp11.23), DXS988 (Xp11.21), DXS101 (Xq21.3), FMR-1 (Xq27.3), and DXYS64 (Xq28). This ring X chromosome is paternally derived since only maternal alleles are inherited at three informative microsatellite loci. Results of FISH and RT-PCR experiments indicate that the XIST locus is missing in the ring X chromosome and not expressed. These data indicated a large deletion of the X chromosome consistent with a small-ring X chromosome with approximate breakpoints near p11.2 and q13. These results are comparable to the observation of others where an atypically severe phenotype has been associated with the presence of an r(X), or small mar(X).

Prader-Willi and Angelman syndromes: diagnosis with a bisulfite-treated methylation-specific PCR method.

The putative promoter region of the SNRPN gene contains a CpG island which is heavily methylated in the maternally derived allele and unmethylated in the paternally derived allele. In patients with Prader-Willi syndrome (PWS) only the methylated allele is present, while in those with Angelman syndrome (AS) only the unmethylated allele is present. The purpose of this paper is to report a polymerase chain reaction (PCR)-based assay to evaluate methylation status of the CpG island of the SNRPN gene and to show that this assay allows rapid diagnosis of PWS and AS. Methylated cytosines in the CpG dinucleotide are resistant to chemical modification by sodium bisulfite. In contrast, bisulfite treatment converts all unmethylated cytosines to uracil. Based on this differential effect, the bisulfite-modified DNA sequence of a methylated allele was successfully distinguished from that of an unmethylated allele using 2 sets of allele-specific primer pairs: a methylated allele-specific primer pair (MET) and an unmethylated allele-specific primer pair (UNMET). Bisulfite-modified DNA from 10 patients with PWS amplified only with the MET pair while modified DNA from 5 patients with AS amplified only with the UNMET pair. Modified DNA from 50 normal unrelated individuals amplified with both primer pairs. In that methylation-specific PCR (MSPCR) can detect all presently testable causes of PWS and AS in a rapid and cost-effective fashion, serious consideration should be given to the use of this test in the initial evaluation of all patients in which PWS or AS is being considered.

Complete hydatidiform mole with a coexistent embryo.

We describe a case of a complete hydatidiform mole with a coexistent embryo. A 19-year-old Hispanic woman presented with an intrauterine pregnancy at 7 weeks gestational age. An ultrasound evaluation at that time showed a gestational sac and fetal heart activity was observed. Four weeks later, subsequent ultrasound study revealed no evidence of an embryo, and a pattern consistent with a hydatidiform mole. Dilation and curettage was subsequently performed that showed a classic hydatidiform mole on histological examination. Chromosome analysis revealed a normal 46,XX karyotype. DNA was extracted from the placental tissue, as well as maternal and paternal blood. Molecular genetic analysis was; performed with four variable number of tandem repeats (VNTR) probes and showed the placental tissue to consist of only paternal DNA with two genomic copies of each allele studied. These findings are consistent with the diagnosis of complete hydatidiform mole and its origin from an empty ovum fertilized by a single sperm. This is the first reported case of a living embryo coexistent with a complete hydatidiform mole documented by genetic analysis.

Congenital adrenal hyperplasia presenting as massive adrenal incidentalomas in the sixth decade of life: report of two patients with 21-hydroxylase deficiency.

Divergent recommendations exist regarding the evaluation of adrenal incidentalomas. Recent data have indicated a prevalence of adrenal tumors of 71% in nonclassical congenital adrenal hyperplasia (CAH) and unmasked heterozygotes. These data expand the differential diagnosis of such incidental tumors and substantially modify the approach to their evaluation. We present two patients, female pseudohermaphrodites with the simple virilizing form of CAH and 21-hydroxylase deficiency, who functioned successfully as married phenotypic males. Both came to medical attention in the sixth decade by virtue of massive adrenal incidentalomas encountered in the evaluation of recurrent urinary tract infections. Each had a 46, XX karyotype, no palpable testes, and markedly elevated baseline levels of 17-hydroxyprogesterone (17-OH Prog) of 6086 ng/dL and 6750 ng/dL. Both responded appropriately to dexamethasone suppression with reduction of 17-OH Prog, androgens and, in the second patient, ACTH to normal or near normal levels. Histologic and autopsy examination of the first patient's tumor and computed tomographic characteristics of the second revealed a benign adenoma and myelolipoma respectively. We extend and confirm previous recommendations that CAH be included in the differential diagnosis of adrenal incidentaloma and that baseline 17-OH Prog. levels be obtained, with ACTH stimulation if necessary, to diagnose the presence of nonclassical CAH.

Unbalanced translocation, t(18;21), detected by fluorescence in situ hybridization (FISH) in a child with 18q- syndrome and a ring chromosome 21.

We report on an 8-year-old girl with minor anomalies consistent with 18q- syndrome and mild developmental delay. Initially cytogenetics showed a terminal deletion of chromosome 21 with mosaicism for a small ring chromosome 21 as the only apparent karyotypic abnormality: mos 45,XX,-21/46,XX,+r(21) (48%/52%). Further studies including FISH and DNA analysis demonstrated a de novo unbalanced translocation of chromosomes 18 and 21 with the likely breakpoints in 18q23 and 21q21.1. Most of 21q was translocated to the distal long arm of one chromosome 18, and this derivative 18 appeared to lack 18q23-qter. The small ring chromosome 21 [r(21)], present in only 52% of the patient's blood lymphocytes, did not appear to be associated with the abnormal phenotype since all 13 chromosome 21 markers that were examined in genomic DNA were present in 2 copies, and the phenotype of the patient was consistent with the 18q- syndrome. The karyotype was reinterpreted as mos 45,XX,-18,-21,+der(18) t(18;21) (q23;q21.1)/46,XX,-18,-21,+der(18) t(18;21) (q23;q21.1), +r(21) (p13q21.1) (48%/52%). These results demonstrate the power of FISH in conjunction with DNA analysis for examination of chromosome rearrangements that may be misclassified by traditional cytogenetic studies alone.

Severe hemophilia A in a female by cryptic translocation: order and orientation of factor VIII within Xq28.

We report studies of a female with severe hemophilia A resulting from a complex de novo translocation of chromosomes X and 17 (46,X,t(X;17)). Somatic cell hybrids containing the normal X, the der(X), or the der(17) were analyzed for coagulation factor VIII (F8C) sequences using Southern blots and polymerase chain reaction. The normal X, always late replicating, contains a normal F8C gene, whereas the der(X) has no F8C sequences. The der(17) chromosome containing Xq24-Xq28 carries a functional G6PD locus and a deleted F8C allele that lacks exons 1-15. Also, it lacks the DXYS64-X locus, situated between the F8C locus and the Xq telomere. These results indicate that a cryptic breakpoint within Xq28 deleted the 5' end of F8C, but left the more proximal G6PD locus intact on the der(17) chromosome. As the deleted segment includes the 5' half of F8C as well as the subtelomeric DXYS64 locus, F8C must be oriented on the chromosome with its 5' region closest to the telomere. Therefore, the order of these loci is Xcen-G6PD-3'F8C-5'F8C-DXYS64-Xqtel. The analysis of somatic cell hybrids has elucidated the true nature of the F8C mutation in the proband, revealing a more complex rearrangement (three chromosomes involved) than that expected from cytogenetic analysis, chromosome painting, and Southern blots. A 900-kb segment within Xq28 has been translocated to another autosome. Hemophilia A in this heterozygous female is due to the decapitation of the F8C gene on the der(17) and inactivation of the intact allele on the normal X.

Spectrum of mutations in CRM-positive and CRM-reduced hemophilia A.

Hemophilia A is due to the functional deficiency of factor VII (FVIII, gene locus F8C). Although half the patients have no detectable FVIII protein in their plasma, the more rare patients (approximately 5%) have normal levels of a dysfunctional FVIII and are termed cross-reacting material (CRM)-positive. More commonly (approximately 45%), patients have plasma FVIII protein reduced to an extent roughly comparable to the level of FVIII activity and are designated CRM-reduced. We used denaturing gradient gel electrophoresis to screen for mutations within the F8C gene of 11 patients (6 CRM-positive, 5 CRM-reduced) and identified 9 different mutations in 9 patients after analyses of all 26 exons, the promoter region, and the polyadenylation site. Six mutations have not been described previously. Five were missense (Ser289Leu, Ser558Phe, Val634Ala, Val634-Met, Asn1441Lys), and the sixth was a 3-bp deletion (delta Phe652). A review of the literature and the assay of FVIII antigen in 5 hemophilia A patients with previously identified missense mutations from this laboratory yielded a total of 20 other unique CRM-reduced and CRM-positive mutations. Almost all CRM-positive/reduced mutations (24/26) were missense, and many (12/26) occurred at CpG dinucleotides. We examined 19 missense mutations for evolutionary conservation using the portions of the porcine and murine F8C sequences that are known, and 18/19 amino acid residues altered by mutation in these patients were conserved. Almost 50% of mutations (11/26) clustered in the A2 domain, suggesting that this region is critical for the function of FVIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of ring chromosome formation in 11 cases of human ring chromosome 21.

We studied the mechanism of ring chromosome 21 (r(21)) formation in 13 patients (11 unique r(21)s), consisting of 7 from five families with familial r(21) and 6 with de novo r(21). The copy number of chromosome 21 sequences in the rings of these patients was determined by quantitative dosage analyses for 13 loci on 21q. Nine of 11 r(21)s, including the 5 familial r(21)s, showed no evidence for duplication of 21q sequences but did show molecular evidence of partial deletion of 21q. These data were consistent with the breakage and reunion of short- and long-arm regions to form the r(21), resulting in deletion of varying amounts of 21q22.1 to 21qter. The data from one individual who had a Down syndrome phenotype were consistent with asymmetric breakage and reunion of 21q sequences from an intermediate isochromosome or Robertsonian translocation chromosome as reported by Wong et al. Another patient, who also exhibited Down syndrome, showed evidence of a third mechanism of ring formation. The likely initial event was breakage and reunion of the short and long arms, resulting in a small r(21), followed by a sister-chromatid exchange resulting in a double-sized and symmetrically dicentric r(21). The phenotype of patients correlated well with the extent of deletion or duplication of chromosome 21 sequences. These data demonstrate three mechanisms of r(21) formation and show that the phenotype of r(21) patients varies with the extent of chromosome 21 monosomy or trisomy.

Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.

Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.

Physical mapping by PFGE localizes the COL3A1 and COL5A2 genes to a 35-kb region on human chromosome 2.

The genes encoding the alpha 1 chain of Type III collagen (COL3A1) and the alpha 2 chain of Type V (COL5A2) collagen have been mapped to the long arm of human chromosome 2. Linkage analysis in CEPH families indicated that these two genes are close to each other, with no recombination in 37 informative meioses. In the present study, DNA probes from the 3' ends of each gene have been physically mapped by pulsed-field gel electrophoresis. The probes recognized 11 macrorestriction fragments in common, ranging from greater than 1000 kb MluI and NotI fragments to a 35-kb SfiI fragment. Therefore, the COL3A1 and COL5A2 genes appear to exist as a gene cluster on chromosome 2. This is the third example of a collagen gene cluster. Other examples include the COL4A1-COL4A2 genes on chromosome 13q and the COL6A1-COL6A2 genes on chromosome 21q. The physical proximity of these genes may indicate common evolution and/or regulation.

In vivo hprt mutant frequencies in T-cells of normal human newborns.

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 X 10(-6) (SD = 0.41 X 10(-6); median value = 0.58 X 10(-6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.

Molecular analyses of in vivo hprt mutations in human T-lymphocytes: IV. Studies in newborns.

In order to characterize in vivo gene mutations that occur during fetal development, molecular analyses were undertaken of mutant 6-thioguanine resistant T-lymphocytes isolated from placental cord blood samples of 13 normal male newborns. These mutant T-cells were studied to define hypoxanthine-guanine phosphoribosyltransferase (hprt) gene structural alterations and to determine T-cell receptor (TCR) gene rearrangement patterns. Structural hprt alterations, as shown by Southern blot analyses, occurred in 85% of these mutant clones. These alterations consisted mostly of deletion of exons 2 and 3. These findings contrast with the 10-20% of gross structural alterations (i.e., those visible on Southern blots) occurring randomly across the entire gene previously reported for T-cell mutants isolated from normal young adults. Iterative analyses of hprt structural alterations and TCR gene rearrangement patterns show that approximately one-third of the newborn derived mutants may have originated as pre- or intrathymic hprt mutations. This too contrasts with previous findings in adults where the background in vivo hprt mutations appeared to originate in postthymic T-lymphocytes.