Detection of gonadal mosaicism in parents of children with Down syndrome

The paper presents results of a revision of data of both conventional chromosome testing and a study of cytogenetic (QFQ) markers in families with Down syndrome. Retrospective analysis of 151 families found eight families with a carrier of gonadal mosaicism. In all cases, the mother was younger than 35 years old. Therefore a prevalence of parental mosaicism in young couples was estimated to be 6.5% (8/123). Conventional diagnostic testing, not followed by analysis of segregation of QHQ markers, would have resulted in a prevalence of only 1%. A comparison of the results ofcytogenetic analysis with those expected using molecular polymorphisms suggests that cytogenetic testing cannot be entirely replaced by molecular testing. A combination of both methods should be applied when gonadal mosaicism is suspected.     

Prenatal diagnosis and characteristics of chromosome aberrations in families with genetic risk

Characteristics of the chromosomal aberrations diagnosed in 959 prenatal tests in the II trimester of pregnancy is presented. Chromosomal aberrations were diagnosed in 33 tests (3.4%). Twenty one out of these aberrations (2.2%) were of labile character. Six aberrations resulted from the parental segregation, translocation or chromosomal inversion. In 12 cases fetus inherited stable aberration from one of parents. It amounted to 1.2% of all tested cases. Chromosomal aberrations were diagnosed in 2.7% cases tested due to the risk related to the mother's age. Half of them was trisomy of chromosome 21. Chromosomal aneuploidy in the progeny of families with a child with the same abnormality was diagnosed in 1.6% of cases. Chromosomal mosaicism was diagnosed in 2.2% of cases including 0.2% of cases with true mosaicism and 1.98% of cases with pseudomosaicism. Incidence and type of the diagnosed chromosomal aberrations coincided with foreseen aberrations for each group of the genetic risk.     

Frequency of somatic and germ-line mosaicism in retinoblastoma: implications for genetic counseling.

Although mosaicism can have important implications for genetic counseling of families with hereditary disorders, information regarding the incidence of mosaicism is available for only a few genetic diseases. Here we describe an evaluation of 156 families with retinoblastoma; the initial oncogenic mutation in the retinoblastoma gene had been identified in these families. In 15 ( approximately 10%) families, we were able to document mosaicism for the initial mutation in the retinoblastoma gene, either in the proband or in one of the proband's parents. The true incidence of mosaicism in this group of 156 families is probably higher than our findings indicate; in some additional families beyond the 15 we identified, mosaicism was likely but could not be proven, because somatic or germ-line DNA from key family members was unavailable. Germ-line DNA from two mosaic fathers was analyzed: in one of these, the mutation was detected in both sperm and leukocyte DNA; in the other, the mutation was detected only in sperm DNA. Our data suggest that mosaicism is more common than is generally appreciated, especially in disorders such as retinoblastoma, in which a high proportion of cases represent new mutations. The possibility of mosaicism should always be considered during the genetic counseling of newly identified families with retinoblastoma. As demonstrated here, genetic tests of germ-line DNA can provide valuable information that is not available through analysis of somatic (leukocyte) DNA.     

Somatic Mosaicism in Hemophilia A: A Fairly Common Event

Mutations in the large gene of clotting factor VIII (FVIII) are the most common events leading to severe human bleeding disorder. The high proportion of de novo mutations observed in this gene raises the possibility that a significant proportion of such mutations does not derive from a single germ cell but instead should be attributed to a germline or somatic mosaic originating from a mutation during early embryogenesis. The present study explores this hypothesis by using allele-specific PCR to analyze 61 families that included members who had sporadic severe hemophilia A and known FVIII gene defects. The presence of somatic mosaicisms of varying degrees (0.2%-25%) could be shown in 8 (13%) of the 61 families and has been confirmed by a mutation-enrichment procedure. All mosaics were found in families with point mutations (8 [25%] of 32 families). In the subgroup of 8 families with CpG transitions, the percentage with mosaicism increased to 50% (4 of 8 families). In contrast, no mosaics were observed in 13 families with small deletions/insertions or in 16 families with intron 22 inversions. Our data suggest that mosaicism may represent a fairly common event in hemophilia A. As a consequence, risk assessment in genetic counseling should include consideration of the possibility of somatic mosaicism in families with apparently de novo mutations, especially families with the subtype of point mutations.     

High rate of mosaicism in tuberous sclerosis complex.

Six families with mosaicism are identified in a series of 62 unrelated families with a mutation in one of the two tuberous sclerosis complex (TSC) genes, TSC1 or TSC2. In five families, somatic mosaicism was present in a mildly affected parent of an index patient. In one family with clinically unaffected parents, gonadal mosaicism was detected after TSC was found in three children. The detection of mosaicism has consequences for genetic counseling of the families involved, as changed risks apply to individuals with mosaicism, both siblings and parents. Clinical investigation of parents of patients with seemingly sporadic mutations is essential to determine their residual chance of gonadal and/or somatic mosaicism, unless a mosaic pattern is detected in the index patient, proving a de novo event. In our data set, the exclusion of signs of TSC in the parents of a patient with TSC reduced the chance of one of the parents to be a (mosaic) mutation carrier from 10% to 2%. In the five families with somatic mosaicism, the parent was given the diagnosis after the diagnosis was made in the child.     

The cri du chat syndrome

Summary Data for 331 cri du chat cases, including 34 Danish probands, are reviewed. The incidence and the prevalence among the mentally retarded population amounted to 1/45,000 and 1.5/1000, respectively. No striking association with prenatal events, parental ages, or birth order could be demonstrated. There was a significant excess of females.Parental translocations were present in slightly more than 10% of the families, while more rare cytogenetic aberrations (mosaicism, rings, and de novo translocations) accounted for less than 10% of all cases. The phenotypically relevant segment has been narrowed down to the midportion of the 5p15 band.Clinical, radiologic, and dermatoglyphic features are summarized and discussed, with special attention to the abnormal cry, which persists in many older probands, and to developmental abnormalities. No obvious correlation could be detected between clinical features and the localization of the deletion.No marker locus has yet been assigned to the short arm of chromosome 5.Treatment and prevention are briefly discussed.     

Parental somatic mosaicism for CNV deletions – A need for more sensitive and precise detection methods in clinical diagnostics settings

To further assess the scale and level of parental somatic mosaicism, we queried the CMA database at Baylor Genetics. We selected 50 unrelated families where clinically relevant apparent de novo CNV-deletions were found in the affected probands. Parental blood samples screening using deletion junction-specific PCR revealed four parents with somatic mosaicism. Droplet digital PCR (ddPCR), qPCR, and amplicon-based next-generation sequencing (NGS) were applied to validate these findings. Using ddPCR levels of mosaicism ranged from undetectable to 18.5%. Amplicon-based NGS and qPCR for the father with undetectable mosaicism was able to detect mosaicism at 0.39%. In one mother, ddPCR analysis revealed 15.6%, 10.6%, 8.2%, and undetectable levels of mosaicism in her blood, buccal cells, saliva, and urine samples, respectively. Our data suggest that more sensitive and precise methods, e.g. CNV junction-specific LR-PCR, ddPCR, or qPCR may allow for a more refined assessment of the potential disease recurrence risk for an identified variant.     

Trisomy 21 Down syndrome

Summary The lymphocyte chromosomes of trisomy 21 Down syndrome patients and their parents in a random series of 374 families were analyzed, the objective being the identification of parental mosaicism. The numbers of parents in whom at least two trisomy 21 cells were detected were seven mothers and three fathers, a frequency of 2.7% of families. Confirmation of mosaicism was by identification of parental transmission of the extra chromosome to the progeny, by repeat chromosome analysis, and/or by the presence of more than one affected child. If to these are added six others in whom only one trisomic cell was detected, but with no other supporting evidence, the frequency could be as high as 4.3%. Differences in parental age at the birth of Down syndrome progeny may be accounted for by differences in frequencies of mosaicism in germ cells and somatic tissue. Mosaicism was found more frequently in the mothers than in the fathers, but more data are required for confirmation of a real difference.     

Germline mosaicism in 4q35 facioscapulohumeral muscular dystrophy (FSHD1A) occurring predominantly in oogenesis

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominantly inherited neuromuscular disorder affecting facial and shoulder girdle muscles with subsequent progression to the pelvic girdle and lower extremities. The major gene involved has been localized to chromosome 4q35 (FSHD1A). The 4q35 DNA marker p13E-11 (D4F104S1) detects a de novo EcoRI DNA rearrangement of < 30 kb in isolated and familial cases. The intrafamilial size of the fragment is constant, inversely correlated with the severity, and directly correlated with the age of onset of the condition. There has been evidence of parental mosaicism in FSHD1A for the D4F104S1 locus. Four female and three male clinically unaffected parents have been described to be carriers of EcoRI fragments of the same size as their affected offspring, but with a markedly less intensive hybridization signal (semi-quantitative evidence). In our total sample of 42 FSHD1A families, we found semi-quantitative evidence of parental D4F104S1 mosaicism in 11 families (EcoRI fragment size range: 12–27 kb). On analysis with adjacent 4q35 probes (D4S163, D4S139), additional qualitative evidence of germline mosaicism could be obtained in two families. In our mosaic families and in the families reported in the literature, a female predominance of mosaicism carriers (13 females versus 5 males) could be noted. In our sample, mosaicism was observed in multigeneration families, in families with isolated cases, and in families with two and three affected children from seemingly unaffected parents. A short EcoRI fragment once having emerged in a mosaicism carrier was found to be transmitted autosomal dominantly to subsequent generations. Of all reported sporadic patients, 19% have a mosaic parent. Finding evidence of parental mosaicism in all our families with more than one affected child of seemingly unaffected parents suggests that there is no autosomal recessively inherited form of FSHD1A. Received: 5 March 1996 / Revised: 14 May 1996     

Human chromosomal heteromorphisms in american blacks

Summary One hundred normal American Blacks (B) were studied by sequential QFQ and RFA banding techniques in order to estimate the type and frequency of heteromorphisms. Color heteromorphisms were classified into one of six colors by RFA and intensity variation into one of five levels by QFQ. The data are compared with a previously studied Caucasian population (C). The frequencies of QFQ and RFA heteromorphisms were significantly higher in the Black than in the Caucasian population. No racial difference was noted for chromosome 21 by QFQ, while RFA demonstrated a clear difference. It is concluded that the maximum characterization of racial differences of human chromosomal heteromorphisms was far greater by RFA than with QFQ. The present study suggests differences in QFQ and RFA heteromorphisms among the two races.     

Correlations between karyotype and phenotype in structural and numerical abnormalities of chromosome 18]

Five cases with different abnormalities of chromosome 18 are described: one case with trisomy 18, two cases with ring 18, one case with partial trisomy 18q and one case with a mosaic 18p-/iso 18q. The karyotypes of the parents were normal. Cytogenetic analysis was performed on PHA stimulated blood lymphocytes. GTG, QFQ, MTX banding techniques were used. Karyotype-phenotype correlations are made. All patients present mental retardation, hypotonia and facial dismorphisms. The different degree of mental retardation and the clinical signs are in relation to the different size of deletions or trisomies of the short or long arm of chromosome 18. In the case with mosaicism 18p-/iso18q the phenotype is determined from the chromosomal abnormality more frequent in the cells (18p-).     

Detection of confined placental mosaicism in trisomy 18 conceptions using interphase cytogenetic analysis

Fluorescence in situ hybridization provides a rapid and accurate technique for detecting chromosomal aneuploidy. It is an excellent method for identifying mosaicism in placental tissues following prenatal diagnosis. Mosaicism, in the form of confined placental mosaicism, occurs im approximately 1%–2% of viable pregnancies studied by chorionic villus sampling at 9–11 weeks of gestation. It has been detected in pregnancies with both diploid and trisomic fetuses and appears to have an important effect on the intrauterine fetal survival. Using both standard cytogenetic analysis and fluorescence in situ hybridization, we have studied 12 placentas from pregnancies with trisomy 18 for the presence of chromosomal mosaicism. These included 2 that were spontaneously aborted, 5 that were terminated after prenatal diagnosis, and 4 that were delivered as either stillborn or liveborn. Significant levels of mosaicism, confined exclusively to cytotrophoblast, were detected in 7 pregnancies. This study demonstrates the usefulness of interphase cytogenetic analysis of uncultured tissues as an alternative method for the detection of mosaicism.     

DNA polymorphism analysis in families with recurrence of free trisomy 21

We used DNA polymorphic markers on the long arm of human chromosome 21 in order to determine the parental and meiotic origin of the extra chromosome 21 in families with recurrent free trisomy 21. A total of 22 families were studied, 13 in which the individuals with trisomy 21 were siblings (category 1), four families in which the individuals with trisomy 21 were second-degree relatives (category 2), and five families in which the individuals with trisomy 21 were third-degree relatives, that is, their parents were siblings (category 3). In five category 1 families, parental mosaicism was detected, while in the remaining eight families, the origin of nondisjunction was maternal. In two of the four families of category 2 the nondisjunctions originated in individuals who were related. In only one of five category 3 families, the nondisjunctions originated in related individuals. These results suggest that parental mosaicism is an important etiologic factor in recurrent free trisomy 21 (5 of 22 families) and that chance alone can explain the recurrent trisomy 21 in many of the remaining families (14 of 22 families). However, in a small number of families (3 of 22), a familial predisposing factor or undetected mosaicism cannot be excluded.     

Cytogenetics and fluorescence in situ hybridization assessment of sex-chromosome mosaicism in Klinefelter's syndrome

A retrospective study was carried out in 152 infertile men to determine the prevalence of sex chromosome abnormalities among non-obstructive azoospermic and severe oligospermic men (n = 51) and to evaluate the feasibility of fluorescence in situ hybridization (FISH) techniques to assess mosaicism in Klinefelter's patients in comparison with conventional cytogenetics. Cytogenetic analysis were performed for 51 infertile men and among 14 chromosomal abnormalities found, nine were compatible with Klinefelter's syndrome. FISH staining with a CEP X/CEP Y probes were performed for Klinefelter's patients and for five of them; testes were biopsied for histopathologic examination. Six Klinefelter's patients showed a non-mosaic 47,XXY and three showed a 47,XXY/46,XY mosaic by G or R banding analysis of 20 cells with a ratio of 17%, 20% and 33%, respectively. FISH analysis confirmed mosaicism in only one patient (the first) in whom a third cells population was found. There was no relationship between the ratios of mosaicism by banding and FISH analysis. Conventional histopathologic findings in five non-mosaic Klinefelter's patients confirm the diagnosis of Sertoli Only Cells syndrome. FISH is recommended in Klinefelter's syndrome to define exactly the cytogenetic statute as mosaic or non-mosaic and then discussing prognosis and decision regarding fertility counseling.     

Germ-line mosaicism in tuberous sclerosis: how common?

Two-thirds of cases of tuberous sclerosis complex (TSC) are sporadic and usually are attributed to new mutations, but unaffected parents sometimes have more than one affected child. We sought to determine how many of these cases represent germ-line mosaicism, as has been reported for other genetic diseases. In our sample of 120 families with TSC, 7 families had two affected children and clinically unaffected parents. These families were tested for mutations in the TSC1 and TSC2 genes, by Southern blotting and by single-strand conformational analysis. Unique variants were detected in six families. Each variant was present and identical in both affected children of a family but was absent in both parents and the unaffected siblings. Sequencing of the variants yielded two frameshift mutations, one missense mutation, and two nonsense mutations in TSC2 and one nonsense mutation in TSC1. To determine which parent contributed the affected gametes, the families were analyzed for linkage to TSC1 and TSC2, by construction of haplotypes with markers flanking the two genes. Linkage analysis and loss-of-heterozygosity studies indicated maternal origin in three families, paternal origin in one family, and either being possible in two families. To evaluate the possibility of low-level somatic mosaicism for TSC, DNA from lymphocytes of members of the six families were tested by allele-specific PCR. In all the families, the mutant allele was detected only in the known affected individuals. We conclude that germ-line mosaicism was present in five families with mutations in the TSC2 gene and in one family with the causative mutation in the TSC1 gene. The results have implications for genetic counseling of families with seemingly sporadic TSC.     

Mosaic SCN1A mutations in familial partial epilepsy with antecedent febrile seizures

SCN1A is the most relevant epilepsy gene. Mutations of SCN1A generate phenotypes ranging from the extremely severe form of Dravet syndrome (DS) to a mild form of generalized epilepsy with febrile seizures plus (GEFS+). Mosaic SCN1A mutations have been identified in rare familial DS. It is suspected that mosaic mutations of SCN1A may cause other types of familial epilepsies with febrile seizures (FS), which are more common clinically. Thus, we screened SCN1A mutations in 13 families with partial epilepsy with antecedent febrile seizures (PEFS+) using denaturing high-performance liquid chromatography and sequencing. The level of mosaicism was further quantified by pyrosequencing. Two missense SCN1A mutations with mosaic origin were identified in two unrelated families, accounting for 15.4% (2/13) of the PEFS+ families tested. One of the mosaic carriers with ~25.0% mutation of c.5768A>G/p.Q1923R had experienced simple FS; another with ~12.5% mutation of c.4847T>C/p.I1616T was asymptomatic. Their heterozygous children had PEFS+. Recurrent transmission occurred in both families, as noted in most of the families with germline mosaicism reported previously. The two mosaic mutations identified in this study are less destructive missense, compared with the more destructive truncating and splice-site mutations identified in the majority of previous studies. This is the first report of mosaic SCN1A mutations in families with probands that do not exhibit DS, but manifest only a milder phenotype. Therefore, such families with mild cases should be approached with caution in genetic counseling and the possibility of mosaicism origin associated with high recurrence risk should be excluded.     

Polymorphism and mosaicism of B chromosome number in Korean field mouse <Emphasis Type="Italic">Apodemus peninsulae</Emphasis> (Rodentia) in the Russian Far East

A karyotype analysis of the Apodemus peninsulae (n = 355) from 41 trapping points from the Russian Far East has allowed us to identify B chromosomes in 87.9% of the animals, 61.7% of which are mosaics. Different levels of variability in the B chromosome numbers have been studied, including both the inter- and intrapopulational, as well as intraindividual variability (mosaicism). It was found that the frequencies of the occurrence of individuals with B chromosomes and mosaicism between different population samples were not constant. The range of the modal B chromosome number variability and variation of the xB index (zero to four; on average, the xB amounted to 1.67) were studied for the first time in different samples and populations of this species. Individuals with the predominant numbers of B chromosome (as a rule, zero to two) were revealed in both groups of animals (with stable and mosaic karyotypes), but the frequency was different in geographical regions. The spectra of B chromosome variability were wider in mosaics (zero to seven) compared to animals with stable karyotypes (zero to four). The importance of this for species of the high frequency of individuals with B chromosomes and with mosaicism has been discussed. The adaptive role of the low number of B chromosomes (one to two), as well as the imbalance of the B chromosome system for the species as a whole is assumed.     

Birth and population prevalence of Duchenne muscular dystrophy in the Netherlands

Summary Mutations causing Duchenne muscular dystrophy (DMD) have a short survival. Therefore, birth and population prevalence are maintained by new mutations. The present inventory was made to estimate the birth and population prevalence rates of DMD in the Netherlands. Seven methods of case identification were used. Data on 496 definite, probable or possible DMD patients born since 1961, or alive on January 1, 1983, were obtained. Several methods gave an estimated ascertainment of more than 95%. The prevalence rate at birth of DMD was estimated at 23.7×10^–5 (14215) male live births (MLB) yearly. The prevalence rate in the male population on January 1, 1983 was 5.4×10^–5 (118496). About 1% of the males in this study may have autosomal recessive Duchenne-like muscular dystrophy. Until now there has been no convincing evidence for geographic differences in DMD prevalence at birth. A list of frequency studies of Duchenne muscular dystrophy is included. The DMD mutation rate calculated by the indirect method is 7.9×10^–5 genes per generation. However, this may well be an over-estimate, as this method does not account for germline mosaicism. Using a modified sex ratio method the proportion of sporadic DMD among all cases was estimated to be 0.106 (range 0–0.332). High frequency of germline mosaicism in DMD is a likely cause for the apparent lack of sporadic cases as found in previous studies, if mutation rates in male and female gametes are equal. Therefore, methods for estimating the proportion of new mutants in DMD should take germline mosaicism into account. The modified sex ratio method allows incorporation of data on germline mosaicism if available.     

Uronic acid content in polysaccharides: Estimation by decarboxylation involving use of gas chromatography

A quantitative method for determining uronic acid content in different polysaccharides has been described. The decarboxylation reaction was run with 57% hydriodic acid (HI) in a small flask attached directly to the gas circuit of a chromatograph. After the reaction was completed, the resultant carbon dioxide was supplied to the chromatographic column. A sample uronic acid (0.5–10.0 μmol) yielded quantitative results with a relative standard deviation of 2%. The contribution of nonuronic materials to the error did not exceed 0.6%. The total analysis time amounted to 50–60 min.     

Frequencies of centromeric heteromorphisms of human chromosomes 3 and 4 as detected by QFQ technique: can they be identified by RFA technique?

One hundred normal Caucasians were studied by sequential QFQ and RFA banding techniques in order to estimate the type and frequency of heteromorphisms in the centromeric regions of chromosome 3 and 4. Intensity variants were classified into 1 of 5 levels of QFQ banding. QFQ intensity heteromorphisms (greater than or equal to level 3) for chromosomes 3 and 4 were 62 and 15 percent respectively. The interrelationship between QFQ and RFA variants were also examined. When the centromere was brilliant by QFQ, it was found that it was deep red by RFA; when it was pale by QFQ, it was light red by RFA. Neverthless, a blind coded study could not pick up these color variants by RFA. QFQ banding showed variations of the centromeric regions of chromosomes 3 and 4 while RFA banding failed to demonstrate it. It was concluded that QFQ is the most useful technique in detecting the different intensity levels in the centromeric regions of chromsomes 3 and 4.