Neocentric small supernumerary marker chromosomes (sSMC)--three more cases and review of the literature.

Here we report on three new patients with neocentric small supernumerary marker chromosomes (sSMC) derived from chromosome 2, 13 and 15, respectively. The sSMC(13) and sSMC(15) had inverted duplicated shapes and the sSMC(2) a ring chromosome shape. All three cases were clinically severely abnormal. A review of the available sSMC literature revealed that up to the present 73 neocentric sSMC cases including these three new cases have been reported. Seven of these cases were not characterized morphologically; in the remainder, 80% had an inverted duplication, 17% a ring and 3% a minute shape. 81% of the reported neocentric sSMC carriers showed severe, 12% moderate and 8% no clinical abnormalities. In summary, we report three more neocentric sSMC cases, provide a review on all up to now published cases, highlight their special characteristics and compare them to centric sSMC.

Molecular karyotyping using an SNP array for genomewide genotyping.

BACKGROUND: Chromosomal imbalances are a major cause of developmental defects as well as cancer and often constitute the key in identification of novel disease related genes. Classical cytogenetic methods are limited in resolution and dependent on highly skilled labour, while methods with higher resolution, based on molecular cytogenetics approaches such as matrix CGH, are not widely available. METHODS: We have developed and evaluated a method we term "molecular karyotyping", using readily available and easy to handle oligonucleotide arrays originally designed for parallel genomewide analysis of over 10,000 SNPs. We show that we can easily and reliably detect unbalanced chromosomal aberrations of various sizes from as little as 250 ng of DNA on a single microarray, based on fluorescence intensity information from clusters of SNPs. RESULTS: We determined the resolution of this method through analysis of 20 trios with 21 previously confirmed subtle aberrations sizing between 0.2 and 13 Mb. Duplications and deletions of at least 5 Mb in size were reliably detectable, but detection of smaller aberrations was dependent on the number of SNPs they contained, thus seven of 10 different deletions analysed, with sizes ranging from 0.2 to 3.7 Mb, were not detectable due to insufficient SNP densitiy in the respective region. CONCLUSIONS: Deduction of reliable cut off levels for array peaks in our series of well characterised patients allows the use of the GeneChip Mapping 10K SNP array for performing rapid molecular karyotyping from small amounts of DNA for the detection of even subtle deletions and duplications with high sensitivity and specificity.

Monosomy 1p36--a recently delineated, clinically recognizable syndrome.

Monosomy 1p36 is a recently delineated contiguous gene syndrome, which is now considered to be one of the most common subtelomeric microdeletion syndromes. We report four unrelated patients with subtle deletions within 1p36 confirmed by high resolution karyotyping and FISH. All exhibited severe psychomotor retardation. Microcephaly, seizures, and visual impairment occurred in three subjects. Results of a first routine karyotyping were unrevealing in three probands. The diagnosis was primarily suggested on the basis of a distinct pattern of facial anomalies in all except the first case. This report illustrates that monosomy 1p36 may be recognized clinically, at least in some patients, whereas the diagnosis is easily missed on routine karyotype.

Glutaric aciduria type III: a distinctive non-disease?

Glutaric aciduria type III is a rare metabolic abnormality leading to persistent isolated glutaric acid excretion. We report the clinical and biochemical phenotypes of three affected children. The first patient is a boy with dysmorphic features and a chromosomal deletion (monosomy 6q26-qter) in whom a persistent glutaric aciduria (500 mmol/mol creatinine, normal <10) was detected during a routine metabolic investigation. The second boy suffered from acute gastroenteritis and hyperthyroidism, when an excessively high urinary glutaric acid excretion was identified (1460 mmol/mol creatinine). The third patient is a girl with constantly elevated glutaric acid in her urine (290 mmol/mol creatinine) but no symptoms of significant disease. In all our patients, glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency), glutaric aciduria type II (multiple acyl-CoA dehydrogenation defect), and secondary forms of glutaric aciduria (for example due to intestinal infections or mitochondrial dysfunction) could be excluded. Loading with the precursor amino acid lysine in all patients as well as with pipecolic acid in the third case led to an increase in urinary glutaric acid excretion, proving the endogenous origin of glutarate. Glutaric aciduria type III (a defect reported to be caused by peroxisomal glutaryl-CoA oxidase deficiency) is our presumptive diagnosis. However, peroxisomal glutaryl-CoA oxidase is not well characterized and no reliable approach for the direct determination of this enzyme is available to us. To our knowledge, in the English language literature only a single patient with glutaric aciduria type III has been described. Our cases reported here confirm the earlier assumption that glutaric aciduria type III is not related to a distinctive phenotype. Glutaric aciduria type III appears to be a rare metabolic abnormality, presumably of peroxisomal metabolism. However, its pathophysiological impact still needs further investigation.

FISH monitoring of 100 courses of human leukemias: the cytogenetic viewpoint.

Interphase fluorescence in situ hybridization (I-FISH) analyses were performed from 2 up to 13 times along the course of 100 human leukemias (36 chronic myeloid leukemias, 38 acute myeloblastic leukemias, 17 acute lymphoblastic leukemias, and 9 additional hematopoietic neoplasias) in order to control clonality, evolution, and disappearance of the basic cytogenetic changes. The relevance of these data to confirm clinical remission or to detect minimal residual disease and/or relapse was evaluated. Fifty-four patients were monitored following hematopoietic stem cell transplantation, and 46 cases after chemotherapy. Various chromosome or aberration specific DNA probes were applied for follow-up in the time frame of 1 month up to 13 years. From the cytogenetic point of view, the aim was to determine the power of resolution of the I-FISH technique in the detection of clinically significant changes in the course of disease and its usefulness in daily routine cyto-genetics as compared with classical cytogenetics. In addition, reliability standards of the various DNA probes should be established. In 75 patients with remissions during the entire period of I-FISH monitoring no conspicuous signal constitution was detected. Of 25 relapses or progresses of disease, which were clinically confirmed, 22 were reliably detected by I-FISH, in only 1 case I-FISH monitoring failed to detect the aberrant clone. In 2 patients conventional karyotype analysis confirmed the relapse by detecting complex chromosomal aberrations, but specific probes for I-FISH confirmation were not available. These data suggest that I-FISH analyses in the follow-up of leukemias is a simple and in most cases sufficiently sensitive and highly reliable way of monitoring the outcome of therapy. It may well serve to close the gap between conventional karyotyping and the highly sensitive molecular techniques.

Deletion 15q24-26 in prenatally detected diaphragmatic hernia: increasing evidence of a candidate region for diaphragmatic development.

Survival of children with congenital diaphragmatic hernia (CDH) is mainly dependent on the extent of lung hypoplasia and the presence of additional congenital anomalies or chromosomal aberrations. A chromosomal deletion 15q25-q26.2 in a fetus with prenatally diagnosed CDH and growth retardation is reported. Despite optimal pre- and neonatal management the baby died shortly after birth. There is increasing evidence that the long arm of chromosome 15, and especially the region 15q24 to 15q26, plays a crucial role in the development of the diaphragm. The finding of a deletion within 15q24-26 in a fetus with CDH has to be considered a predictor of poor prognosis. It is of utmost interest for proper parental counselling to search in fetuses with CDH for subtle chromosomal lesions paying special attention to chromosome 15q.

The cytogenetic view of standard comparative genomic hybridization (CGH): deletions of 20q in human leukemia as a measure of the sensitivity of the technique.

BACKGROUND: The limits of the resolving power of comparative genomic hybridization (CGH) have been given as 10-20 Mbp if at least 50% of the studied neoplastic cell population carried the corresponding aberration. MATERIAL AND METHODS: Genomic DNA of five cases of hematologic neoplasias, in all of which--among other anomalies--deletions of different size of chromosome 20q were found by GTG banding and confirmed by FISH analyses, was subjected to CGH. RESULTS: CGH revealed four types of del(20q), and, in addition, detected a tiny terminal del(3p) in one of the cases. The size of the smallest deleted segment, clearly visible by eye on the CGH metaphase image, was estimated to range between 5 and 7 Mbp. CONCLUSION: Visual determination was shown to have a stronger resolving power in CGH than software used for the analysis in one case, while in another one, the results obtained from the ratio profiles would have been considered insignificant without the knowledge of the hybridization pattern on the corresponding CGH metaphase images. The potential of the standard CGH technique not only to detect, but visualize small segmental aneusomies as well, suggests that its resolution actually mirrors the resolution of banding techniques.

First known microdeletion within the Wolf-Hirschhorn syndrome critical region refines genotype-phenotype correlation.

Deletions within HSA band 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which comprises mental retardation and developmental defects. A WHS critical region (WHSCR) of approximately 165 kb has been defined on the basis of 2 atypical interstitial deletions; however, genotype-phenotype correlation remains controversial, due to the large size of deletion usually involving several megabases. We report on the first known patient with a small de novo interstitial deletion restricted to the WHSCR who presented with a partial WHS phenotype consisting only of low body weight for height, speech delay, and minor facial anomalies; shortness of stature, microcephaly, seizures and mental retardation were absent. The deletion was initially demonstrated by FISH analysis, and breakpoints were narrowed with a "mini-FISH" technique using 3-5 kb amplicons. A breakpoint-spanning PCR assay defined the distal breakpoint as disrupting the WHSC1 gene within intron 5, exactly after an AluJb repeat. The proximal breakpoint was not found to be associated with a repeated sequence or a known gene. The deletion encompasses 191.5 kb and includes WHSC2, but not LETM1. Thus, manifestations attributable to this deletion are reduced weight for height, minor facial anomalies, ADHD and some learning and fine motor deficiencies, while seizures may be associated with deletions of LETM1.

Comparative genomic hybridization-aided unraveling of complex karyotypes in human hematopoietic neoplasias.

The information obtained by conventional cytogenetics (CC) in human leukemias is sometimes limited, in particular by complex karyotypes with many marker chromosomes. While CC is restricted to metaphases with a good quality, interphase fluorescence in situ hybridization (I-FISH) is also capable of analyzing specific anomalies in the interphase nuclei. Comparative genomic hybridization (CGH) gives additional information about the imbalanced karyotype changes in the whole genome. The aim of this study was to assess the contribution of CGH to the unraveling of complex GTG karyotypes, which are difficult to evaluate by banding analysis, and to compare these results with those by CC and FISH. Thirteen bone marrow samples and one sample obtained from peripheral blood of 13 leukemia patients were examined by CC, FISH and CGH. The GTG banding analysis showed complex karyotypes with many marker chromosomes. The most frequent abnormalities were numerical and structural aberrations on chromosomes 5 and 7. In 12 of the 14 samples, the CGH analysis was able to detect chromosomal imbalances with losses of material on chromosome 5 and 7 as the most frequent aberrations. In all 14 samples, additional FISH analyses were performed. For most of the studied neoplasias, a close correlation between CC, FISH and CGH data was observed. CGH was considerably helpful in adding additional information to classical karyotyping, if the low quality or number of metaphases was insufficient for a reliable CC analysis. Even in cases where whole chromosome painting could be applied, it added information on the breakpoints of the observed rearrangements. In only 2 of the studied 14 samples, neither CGH nor I-FISH could improve the result of karyotyping. CGH, nevertheless, can be regarded as a powerful additional technique in leukemias with unsuccessful CC, incomplete, or complex karyotypes with many marker chromosomes. A systematic analysis by three techniques such as CC, FISH and CGH guarantees an optimal genetic characterization of the neoplasias.

Prenatal diagnosis of heterokaryotypic mosaic twins discordant for fetal sex.

The presence of a monozygotic twin gestation with discordant sex of the twins is a very rare constellation, which is referred to as heterokaryotypic monozygotic pregnancy. This constellation can develop either due to a chromosomal aberration after twinning or is - as in the following case - due to a mitotic error before twinning and an unequal distribution of mosaicism in both embryos. So far the diagnosis of heterokaryotypic monozygotic pregnancy has always been made postnatally, with only one exception (Gonsoulin et al., 1990). In this case we suspected the presence of monozygotic twins ultrasonically because of the chorionic and amniotic membrane characteristics. Surprisingly the sex of the fetuses was discrepant. As one of them had hydrops and a structural heart defect, we carried out an amniocentesis, which revealed mosaicism [45,X/46,X,i(Y)(p10)] of both fetuses. The female fetus with a predominant 45,X set of chromosomes and the typical intrauterine signs of the Ullrich-Turner syndrome (massive hygroma colli, hydrops fetalis and multiple cardiac defects) died during the 25th week of gestation due to cardiac decompensation. The other fetus appeared to be male with a predominance of a 46,X,i(Y)(p10) set of chromosomes and was born a few days after the intrauterine death of the hydropic fetus. In conclusion, our observation shows that ultrasonic evidence of discordant fetal sex in twins does not necessarily exclude monozygosity.

Delimiting the use of comparative genomic hybridization in human myeloid neoplastic disorders.

Hematopoietic disorders can be used as a suitable tool of additional information on the actual resolving power of comparative genomic hybridization (CGH). Therefore, CGH examination was performed of DNA extracted from 23 acute and 15 chronic myeloproliferative disorders which had just been analyzed using classical cytogenetic techniques. In nearly all cases CGH analysis was repeated with reversely labeled probes. A Zeiss axioplan microscope was equipped with the ISIS 3 system for photometric evaluation of the CGH data. A main group was selected of 34 cases showing karyotypic mosaics when routinely diagnosed by classical cytogenetics. The grade of mosaicism was basically determined from the classical cytogenetic analysis and was additionally defined examining target anomalies by I-FISH analysis in 28 of the cases. The second group included 23 cases with deletions, and in 1 case another informative genomic imbalance could be analyzed. Every target anomaly irrespective of its type could be detected in all cases with an affected cell population equalling or exceeding about 25%, but in none was it below 23%. This value was the lowest and was found in a case, with CGH-detected 20q deletion. The smallest deletions of two bands on 20q which could visually be detected by CGH were estimated in the range of 5-7 Mb. CGH was also suitable to detect imbalances which were not clearly detected by routine cytogenetics. Reverse labelling, performed in nearly all cases, confirmed the result of the original CGH analysis. These data not only document the readiness and reliability of CGH studies on human leukemia, but also further contribute to a clearer definition of the limits of the resolving power of this technique.

FISH mapping of the sex-reversal region on human chromosome 9p in two XY females and in primates.

Accumulating evidence suggests that haploinsufficiency of a dosage-sensitive gene(s) in human chromosome 9p24.3 is responsible for the failure of testicular development and feminisation in XY patients with monosomy for 9p. We have used molecular cytogenetic methods to characterise the sex-reversing 9p deletions in two XY females. Fluorescence in situ hybridisation (FISH) with YACs from the critical 9p region containing an evolutionarily conserved sex-determining gene, DMRT1, is a very fast and reliable assay for patient screening. Comparative YAC mapping on great ape and Old and New World monkey chromosomes demonstrated that the critical region was moved from an interstitial position on the ancestral primate chromosome to a very subtelomeric position in chimpanzee and humans by a pericentric inversion(s). Pathological 9p rearrangements may be the consequence of an evolutionary chromosome breakpoint in close proximity to the sex-reversal region.

Defective sexual development in an infant with 46, XY, der(9)t(8;9)(q23.1;p23)mat.

UNLABELLED: We report on a male infant with ambiguous genitalia (scrotal hypospadias, sinus urogenitalis) trisomic for 8q23-ter and monosomic for 9p23-ter, who shared craniofacial and other abnormalities with either phenotype. Gonadal histology was nearly normal for age. Normal endocrinological findings and exclusion of mutations in SRY, androgen receptor and alpha-reductase genes point to supplementary gene(s) located in 9p2305-ter, haplo-insufficiency (by deletion) of which is expected to cause defective male morphogenesis. CONCLUSION: This observation lends further support to the hypothesis that genetic factors are located at 9p23-ter which are involved in normal sex determination.

Further observations of true mosaic trisomy 17 ascertained in amniotic fluid cell cultures.

Three new cases of true mosaic trisomy 17 (MT17) were diagnosed in amniotic fluid cells. Postnatal chromosome analysis from lymphocytes did not confirm the trisomic cell line, and follow-up studies showed normal psycho-motor development of the children, in one case up to the age of 4 1/2 years. We suggest that there are similarities between MT17 and MT20, in which the majority of pregnancies result in deliveries of healthy babies.

Incidence and significance of 22q11.2 hemizygosity in patients with interrupted aortic arch.

Interruption of the aortic arch (IAA) is a severe malformation of the heart with known association to DiGeorge syndrome (DGS) and 22q11.2 hemizygosity. The aim of this study was to establish incidence and significance of 22q11.2 hemizygosity in an unbiased sample of patients with IAA. All 15 children with IAA who were referred to our hospital in a 3-year period were tested by chromosome and fluorescence in situ hybridization (FISH) analysis with the probes D22S75, Tuplel, and cHKAD26 and by a set of 10 simple tandem repeat polymorphic (STRP) markers. In nine of 11 children with IAA type B, 22q11.2 hemizygosity was demonstrated by FISH and STRP analysis, but in none of the four children with type A. In all but one child, deletion size was approximately 3 Mb. The girl with the smaller deletion of approximately 1.5 Mb differed because of an Ullrich-Turner syndrome-like phenotype and severe T-cell defect. Additionally, in one patient with phenotypic signs of DGS, a small deletion distal to the known DGS region containing the marker D22S308 was suspected by STRP analysis. One deletion was shown to be inherited from a healthy father and one IAA type A recurred in a sib. T-cell anomalies were evident in eight of the nine children with classical deletion, five of whom suffered also from hypoparathyroidism. With respect to cause and clinical course, IAA type A and B were shown to represent different entities. This study showed that variable symptoms of 22q11.2 hemizygosity may cluster.

Interstitial deletion del(3)(p12p21) in a malformed child subsequent to paternal paracentric insertion (or intraarm shift) 46,XY, ins(3)(p24.1p12.1p21.31).

We report on a malformed stillborn with deletion 3p subsequent to direct paracentric insertion (intraarm shift) in the normal father which had been first mistaken for paracentric inversion. The corrected diagnosis was supported by FISH of mapped markers on metaphase chromosomes. In addition we looked for recombinants in sperm. This observation reminds similar cases that had been considered exceptions to the expected meiotic recombination of paracentric inversions and points to a cytogenetic pitfall. Published deletions and paracentric inversions in 3p are briefly quoted.

Monozygotic twins concordant for Cayler syndrome.

Deletions within chromosome band 22q11.2 are associated with a variety of conditions, although a simple genotype-phenotype correlation has not been established so far. Environmental factors, chance events, or a second hit theory were supported by two observations of monozygotic twins with 22q11.2 deletions and discordant phenotypes [Goodship et al., J Med Genet 1995;32:746-748; Fryer, J Med Genet 1996;33:173]. We present monozygotic twins concordant for 22q11.2 deletion and Cayler syndrome, favoring the view that there exists a predominant genetic determination of the del 22q11.2 phenotype. As these twins are diamniotic and dichorionic, they may offer a more reliable insight in genetic phenotype determination than the other published, probably monochorionic, twins who may have a discordant malformation by twinning itself.

Brachydactyly in a child with duplication-deficiency subsequent to t(15;20)(q25.2;p12.2)mat. Candidate regions on one or both chromosomes?

We report a child with a duplication-deficiency subsequent to t(15;20)(q25.2;p12.2), transmitted in at least 5 generations, who showed features of 15q- syndrome. We speculate that brachydactyly--most likely because of brachymesophalangism--is a feature of the phenotype of this chromosomal aberration and points to candidate gene(s) in this region. A similar brachydactyly was, however, reported with dup(20p1-pter).