Mikael Donnér

Chromosomal aberrations in a consecutive series of childhood rhabdomyosarcoma

BACKGROUND AND PROCEDURE During a 13-year period, 22 children were treated for rhabdomyosarcoma (RMS). In 18 of these patients chromosome analysis was attempted on material from tumor biopsies, fine needle aspiration biopsies and/or bone marrow samples. RESULTS Clonal chromosome aberrations were detected in 14 cases; 7 of 9 embryonal RMS, 6 of 8 alveolar RMS and in the single case of pleomorphic RMS cytogenetic failures were more frequent in fine needle aspiration biopsies than in tumor biopsies. The characteristic t(2;13) translocation was seen in 2 alveolar RMS but not in any of the other subtypes. In 3 of the embryonal RMS hyperdiploid or hypertetraploid karyotypes with few or no structural rearrangements were seen. In all 3 cases the clinical course was relatively benign, suggesting that certain karyotypic patterns in RMS may be of prognostic significance. CONCLUSIONS Our results add to the evidence that cytogenetic analysis should be an integral part of the diagnostic examinations of children with RMS.

Cytogenetic aberrations in Ewing sarcoma: are secondary changes associated with clinical outcome?

BACKGROUND Ewing sarcoma is associated with a nonrandom pattern of primary and secondary chromosomal aberrations. Whereas the finding of rearrangements of chromosome 22, usually in the form of a balanced translocation t(11;22)(q24;q12), is important diagnostically, nothing is known about the potential prognostic impact of the secondary chromosomal aberrations. PROCEDURE During a 1 3-year-period, short-term cultured tumor samples from 21 children and young adults with Ewing sarcoma were cytogenetically analyzed successfully. RESULTS Clonal chromosome aberrations were detected in 18 patients, 17 of whom had the characteristic t(11;22)(q24;q12) or variants thereof. The most frequent secondary change was +8, followed by +12, +2, +5, +9, +15, and gain of material from the long and short arms of chromosome 1. The only recurrent secondary change that was restricted to tumors from the ten patients that were dead at latest follow-up was gain of 1q material. Furthermore, all three patients with tumors with chromosome numbers over 50 had died, and the only patient with a tumor karyotype lacking chromosome 22 rearrangement was alive without evidence of disease. CONCLUSIONS These data and previously published results indicate that the karyotypic pattern not only may be of diagnostic significance but also may be important prognostically.

Type IIB von Willebrand's disease with probable autosomal recessive inheritance and presenting as thrombocytopenia in infancy.

von Willebrands disease (vWD) is a congenital bleeding disorder that exists in two main forms. In the classic form, type I, the concentration of the von Willebrand factor (vWF) in plasma is decreased. In type II vWD, the vWF is structurally altered. Type II can be further divided into at least six subtypes (A, B, C, D, E and F). In type IIB the vWF, in contrast to other variants of vWD, shows an increased affinity for platelets. IIB vWD is generally believed to be inherited in an autosomal dominant manner. We describe two families with three affected children in whom an autosomal recessive inheritance is more likely. Thrombocytopenia, constant or variable, was present from early infancy in all three cases. Type IIB vWD should thus be included in the differential diagnosis of congenital thrombocytopenia.

Type iib von willebrand's disease gene mutations and clinical presentation in nine families from denmark germany and sweden

Summary Type IIB of von Willebrands disease (vWD) is a variant in which the structurally abnormal von Willebrand factor (vWF) shows an increased affinity for the platelet vWF receptor, glycoprotein Ib (GPIb). This may sometimes give rise to platelet aggregation and thrombocytopenia in vivo. In 20 patients from nine unrelated families with type IIB vWD from Denmark, Germany and Sweden we studied the molecular defect by amplification and direct sequencing of parts of exon 28 which encode for the vWF domain that interacts with platelet GPIb. Three different point mutations were identified one of which has not previously been reported. Fifteen patients from five families were heterozygous for the Arg543Trp substitution. The mutation had occurred independently in all five families and in two of them represented a de novo mutation. In one of these families the father, though asymptomatic and with normal laboratory test results, carried the mutation in heterozygous form. In three families, four affected members were found to be heterozygous for the Arg543°Cys substitution. The mutations were of different origin at least in two of the families. The third substitution, Val554Leu, which has not previously been described, was found in one patient and was due to a de novo mutation. In most of the patients spontaneous thrombocytopenia had been recorded on at least one occasion. Five of the patients with the Arg543Trp substitution and the one with the Val555Leu substitution had all had bleeding associated with thrombocytopenia in the neonatal period of early infancy.

An HphI‐polymorphism in exon 28 of the von Willebrand factor gene, and its frequency among patients with various forms of von Willebrand's disease

Summary. Besides having a large number of restriction fragment length polymorphisms (RFLP) the von Willebrand factor (vWF) gene contains several sequence polymorphisms in the coding regions. Eight nucleotide substitutions have been reported in two or more independent cDNA clones. Four of them give rise to amino acid substitutions, two of which are in the mature vWF subunit (at positions 26 and 709). We have investigated a previously suggested putative alaninethreonine polymorphism at position 618 of the mature subunit in normal subjects and patients with various types of von Willebrands disease (vWD). The codon for amino acid 618 is located in exon 28, which encodes several important vWF functional domains. We amplified the whole exon 28 and parts of it by polymerase chain reaction (PCR) and distinguished gene from pseudogene sequences. The alanine→lthreonine (G→A) substitution was studied with restriction enzyme cleavage of the products, since it creates a new HphI site. Moreover, in two individuals we confirmed the polymorphism by cDNA sequencing. In 23 normals the frequencies of the h‐ (Ala) and the h+ (Thr) alleles were 0·50/0·50. In eight patients with type III vWD from seven different families, the h– allele was present in 13 of 16 genes, but whether this signifies a common mutation in some of the patients is not known. In types I and II. both alleles were present in roughly similar proportions. Owing to the high frequency of heterozygosity, the polymorphism should prove useful as an aid in genetic counselling.

Childhood acute lymphoblastic leukaemia with ider(21)(q10)t(12;21)(p12;q22): a new recurrent abnormality showing ETV6/CBFA2 fusion

The cytogenetically unidentifiable t(12;21) (p12;q22), resulting in ETV6/CBFA2 fusion, is the most frequent chromosomal aberration in childhood acute lymphoblastic leukaemia (ALL). We report a variant, ider(21) (q10)t(12;21)(p12;q22), which was shown to contain double ETV6/CBFA2 fusions by fluorescence in situ hybridization. This is the second case of such an ider(21) in childhood ALL, suggesting that it is a new recurrent abnormality. Since the ider(21) is cytogenetically indistinguishable from i(21)(q10) and idic(21)(p11), changes associated with similar clinical features as the t(12;21), i.e. pre‐B‐cell ALL and age 1–10 years, we suggest that all ALL displaying these changes should be tested for ETV6/CBFA2 fusion transcript.

Two new candidate mutations in type IIA von Willebrand's disease (ARG834→GLY, GLY846→ARG) and one polymorphism (TYR821→CYS) in the A2 region of the von Willebrand factor

Abstract: Recently, several von Willebrand factor gene mutations resulting in type IIA von Willebrands disease have been reported. We examined 8 patients from Sweden and Denmark with this phenotype and found two new candidate mutations and a hitherto unknown amino acid polymorphism. One patient had a de novo occurring mutation resulting in substitution of glycine for arginine 834. Previous reports have demonstrated conversion of arginine 834 to tryptophan or glutamine in IIA patients. A 2nd patient had a G(4825)→A transition, substituting arginine for glycine 846. The transition produces a sequence congruent with that of the pseudogene but several lines of evidence indicate that a sequencing error due to influence by the latter could be excluded. The remaining 6 patients had one of the earlier described substitution mutations: Ser743→Leu and Ile865→Thr. In addition, two sequence variations not linked to the phenotype were found, namely Tyr821→Cys and Val802→Leu.

An Arg545→ Cys545 substitution mutation of the von Willebrand factor in type IIB von Willebrand's disease

Abstract: Type IIB is a special variant of von Willebrands disease, characterized by an abnormal von Willebrand factor which shows an increased interaction with platelets. This interaction sometimes causes platelet aggregation and thrombocytopenia in vivo. It involves the glycoprotein‐Ib (GPIb) receptor on platelets and corresponding GPIb‐binding sites in the von Willebrand factor. We here demonstrate a C ± T mutation at codon 1308 of the von Willebrand factor gene in 2 related patients with IIB von Willebrands disease. The transition gives rise to a substitution of arginine by cysteine at position 545 of the mature von Willebrand factor subunit. This position is close to the GPIb‐ as well as the collagen‐ and heparin‐binding domains of the von Willebrand factor. The mutation may change the conformation of the molecule in this region and activate the GPIb‐binding domain, which is normally not exposed in the von Willebrand factor of circulating blood.

Clonal karyotypic evolution in a pediatric neurofibrosarcoma

A retroperitoneal neurofibrosarcoma infiltrating the spine of a 2-year-old boy was investigated cytogenetically three times over a 5-month period. The first sample, from a diagnostic fine-needle aspiration biopsy, had a supernumerary i(1)(q10) as the sole clonal aberration; two cells showed monosomy 18 in addition to the isochromosome. The second sample, obtained at tumor resection 3 weeks later, had the karyotype 47,XY, +i(1)(q10), -18, +21/45,XY, -18. After 5 months, a large local recurrence was resected. The chromosome analysis showed further clonal evolution: 45,XY, +1,der (1;11)dic(1;11)(q44;q13)i(1)(q10), inv(6)(p21q12), -17. The findings indicate that formation of i(1)(q10) and loss of chromosome 18 may be early genetic events in neurofibrosarcoma development.