Jörg Seidel

DNA Ligase IV Mutations Identified in Patients Exhibiting Developmental Delay and Immunodeficiency

DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS). Some of the mutations identified in the patients directly disrupt the ligase domain while others impair the interaction between DNA ligase IV and Xrcc-4. Cell lines from the patients show pronounced radiosensitivity. Unlike NBS cell lines, they show normal cell cycle checkpoint responses but impaired DNA double-strand break rejoining. An unexpected V(D)J recombination phenotype is observed involving a small decrease in rejoining frequency coupled with elevated imprecision at signal junctions.

Homologous sequences at human chromosome 9 bands p12 and q13-21.1 are involved in different patterns of pericentric rearrangements

A thorough study of the heterochromatin organisation in the pericentromeric region and the proximal long (q) and short (p) arms of human chromsome 9 (HSA 9) revealed homology between 9p12 and 9q13-21.1, two regions that are usually not distinguishable by molecular cytogenetic techniques. Furthermore, the chromosomal regions 9p12 and 9q13-21.1 showed some level of homology with the short arms of the human acrocentric chromosomes. We studied five normal controls and 51 clinical cases: 48 with chromosome 9 heteromorphisms, one with an exceptionally large inversion and two with an additional derivative chromosome 9. Using fluorescence in situ hybridisation (FISH) with three differentially labelled chromosome 9-specific probes we were able to distinguish 12 heteromorphic patterns in addition to the most frequent pattern (defined as normal). In addition, we studied one inversion 9 case with the recently described multicolour banding (MCB) technique. Our results, and previously published findings, suggest several hotspots for recombination in the pericentromeric heterochromatin of HSA 9. They also demonstrate that constitutional inversions affecting the pericentromeric region of chromosome 9 carry breakpoints located preferentially in 9p12 or 9q13-21.1 and less frequently in 9q12.

Next-generation sequencing in X-linked intellectual disability

X-linked intellectual disability (XLID) is a genetically heterogeneous disorder with more than 100 genes known to date. Most genes are responsible for a small proportion of patients only, which has hitherto hampered the systematic screening of large patient cohorts. We performed targeted enrichment and next-generation sequencing of 107 XLID genes in a cohort of 150 male patients. Hundred patients had sporadic intellectual disability, and 50 patients had a family history suggestive of XLID. We also analysed a sporadic female patient with severe ID and epilepsy because she had strongly skewed X-inactivation. Target enrichment and high parallel sequencing allowed a diagnostic coverage of >10 reads for ~96% of all coding bases of the XLID genes at a mean coverage of 124 reads. We found 18 pathogenic variants in 13 XLID genes (AP1S2, ATRX, CUL4B, DLG3, IQSEC2, KDM5C, MED12, OPHN1, SLC9A6, SMC1A, UBE2A, UPF3B and ZDHHC9) among the 150 male patients. Thirteen pathogenic variants were present in the group of 50 familial patients (26%), and 5 pathogenic variants among the 100 sporadic patients (5%). Systematic gene dosage analysis for low coverage exons detected one pathogenic hemizygous deletion. An IQSEC2 nonsense variant was detected in the female ID patient, providing further evidence for a role of this gene in encephalopathy in females. Skewed X-inactivation was more frequently observed in mothers with pathogenic variants compared with those without known X-linked defects. The mutation rate in the cohort of sporadic patients corroborates previous estimates of 5–10% for X-chromosomal defects in male ID patients.

Brachytelephalangic dwarfism due to the loss of ARSE and SHOX genes resulting from an X;Y translocation

Here we report an 8‐year‐old male patient who had mesomelic shortening of forearms and legs, brachytelephalangia and ichthyotic skin lesions. Chromosomal analysis showed an X;Y translocation involving the short arm of the X chromosome (Xp). Fluorescence in situ hybridization (FISH) and molecular studies localized the breakpoints on Xp22.3 in the immediate vicinity of the KAL gene demonstrating deletions of steroid sulfatase (STS), arylsulfatase E (ARSE), and short stature homeo box (SHOX) genes. It was suspected that the patient was suffering from chondrodysplasia punctata because of a loss of the arylsulfatase E (ARSE) gene. However, no stippled epiphyses were to be seen in the neonatal radiograph. Interestingly, this patient is the first case with a proven loss of the ARSE gene without chondrodysplasia punctata, assuming that chondrodysplasia punctata is not an obligatory sign of ARSE gene loss. Brachytelephalangia was the only result of ARSE gene deletion in this case. 
The patients mother also had dwarfism and showed Madelung deformity of the forearms. She was detected as a carrier of the same aberrant X chromosome. The male patient did not show Madelung deformity, demonstrating that Lerri–Weill syndrome phenotype may be still incomplete in children with SHOX gene deletion. The wide clinical spectrum in the male and the Leri–Weill phenotype in his mother are the results of both a deletion involving several sulfatase genes in Xp22.3 and the SHOX gene located in the pseudoautosomal region. Nevertheless, there is no explanation for the absence of chondrodysplasia punctata despite the total loss of the ARSE gene. 
Further studies are necessary to investigate genotype/phenotype correlation in cases with translocations or microdeletions on Xp22.3, including the ARSE and the SHOX gene loci.

Microdeletion 4p16.3 in three unrelated patients with Wolf-Hirschhorn syndrome

Wolf-Hirschhorn syndrome (WHS) is a multiple malformation syndrome caused by partial monosomy of 4p16.3. Pitt-Rogers-Danks syndrome, first thought to be a distinct entity, is a similar condition associated with a microdeletion overlapping the WHS critical region. In this paper we evaluate three WHS patients showing a microdeletion of 4p and remarkable development with respect to the clinical spectrum of WHS.

A multiple translocation event in a patient with hexadactyly, facial dysmorphism, mental retardation and behaviour disorder characterised comprehensively by molecular cytogenetics. Case report and review of the literature

We report a 13-year-old female patient with multiple congenital abnormalities (microcephaly, facial dysmorphism, anteverted dysplastic ears and postaxial hexadactyly), mental retardation, and adipose-gigantism. Ultrasonography revealed no signs of a heart defect or renal abnormalities. She showed no speech development and suffered from a behavioural disorder. CNS abnormalities were excluded by cerebral MRI. Initial cytogenetic studies by Giemsa banding revealed an aberrant karyotype involving three chromosomes, t(2;4;11). By high resolution banding and multicolour fluoresence in-situ hybridisation (M-FISH, MCB), chromosome 1 was also found to be involved in the complex chromosomal aberrations, confirming the karyotype 46,XX,t(2;11;4).ish t(1;4;2;11)(q43;q21.1;p12-p13.1;p14.1). To the best of our knowledge no patient has been previously described with such a complex translocation involving 4 chromosomes. This case demonstrates that conventional chromosome banding techniques such as Giemsa banding are not always sufficient to characterise complex chromosomal abnormalities. Only by the additional utilisation of molecular cytogenetic techniques could the complexity of the present chromosomal rearrangements and the origin of the involved chromosomal material be detected. Further molecular genetic studies will be performed to clarify the chromosomal breakpoints potentially responsible for the observed clinical symptoms. Conclusion: this report demonstrates that multicolour-fluorescence in-situ hybridisation studies should be performed in patients with congenital abnormalities and suspected aberrant karyotypes in addition to conventional Giemsa banding.

Conspicuous GTG‐banding results of the centromere‐near region can be caused by alphoid DNA heteromorphism

To the Editor: It is a well-known fact that heteromorphisms of the alphoid DNA can lead to size variations of the centromeric region. When applying fluorescence in situ hybridization (FISH) using fluorescencelabeled alphoid probes, the signals obtained can vary among ‘absent’, ‘small’, ‘medium’, ‘large’ and ‘very large’, as shown in two previous reports published in Clinical Genetics (1, 2) and in another report of Verma et al. (3). The first two classes of variations (‘absent’ and ‘small’) may lead to false-positive results in interphase cytogenetics, i.e. monosomy of the corresponding chromosome is suspected in tumor-cytogenetic (4) or pre-natal diagnostics (5, 6). Conversely, very large heteromorphic patterns are detected in GTG-banding analysis as abnormal patterning of metaphase chromosomes (7–11). We report on three new cases with conspicuous GTG-banding results of the centromere-near region, initially thought to be centromere-near duplications or pericentric inversions, which were found to be caused by ‘large’ alphoid DNA repeats. The alphoid region on the corresponding homologous chromosomes was ‘medium’ sized. The real character of the suspected chromosomal rearrangements was initially identified by chance (see below for case 1) or after testing other FISH probes previous to alphoid DNA (case 2). Case 1 was a newborn boy with clinical signs resembling CATCH22. However, FISH analysis did not show a microdeletion in 22q11.2. According to GTG analysis, a small centromere-near duplication in 15q11.2 was suspected (Fig. 1a). Therefore, a commercial probe for this region (LSI Prader-Willi/Angelman Region Probe SNRPN; Vysis/Abbot, Wiesbaden-Delkenheim, Germany) was applied. No duplication was detectable by the SNRPN probe. However, the centromeric probe for chromosome 15 (cep 15 – D15Z1), which is included into the commercial probe set as a control, gave a ‘medium’ signal on the normal chromosome 15 and a ‘large’ signal on the chromosome 15 suspected to have a duplication (Fig. 1a). Thus, the real character of the conspicuous GTGbanding result was solved unexpectedly by a probe used as an internal control. One similar case has been reported previously (12). Case 2 was a 34-year-old man referred for cytogenetic analysis because of a subfertility problem. According to GTG banding, a pericentric inversion in one chromosome 4 was suggested (Fig. 1b). FISH, using partial chromosome painting probes specific for chromosomal arms 4p and 4q, respectively, did not provide any evidence for such an inversion (result not shown). Therefore, according to the experience with case 1, a probe specific for the alpha satellite DNA of chromosome 4 (cep 4; Vysis) was applied and revealed a ‘medium’ signal on the normal chromosome 4 and a ‘large’ signal on the chromosome 4 thought to have an inversion (Fig. 1b). A 10-year-old girl (case 3) was analyzed cytogenetically because of growth retardation and the

X-linked forms of chondrodysplasia punctata: Re-description of two cases originally reported by Professor Erich Hässler in 1940 and the knowledge six decades later

Six decades ago, Professor Erich Hassler, one of the oldest still living European pediatricians who celebrated his 103 t h birthday in April 2002, described two patients with chondrodysplasia punctata. He performed profound clinical, radiological and histological studies of a female neonate with severe X-chromosomal dominant chondrodysplasia punctata type Conradi-Hunermann and investigated an 8-year-old male with a probably X-linked recessive form of chondrodysplasia punctata. He compared these patients with 7 previously described cases also accompanied by stippled epiphyses. To honor the personality of Professor Hassler, both cases were repeated again with his consent. These case reports are connected with the recent knowledge about the molecular background of chondrodysplasia punctata. The X-linked types of chondrodysplasia have been distinguished from other diseases also characterized by stippled epiphyses.

Insulin-like growth factor serum concentrations reflect insufficient growth in a hypoplastic infant with partial trisomy 9q in the 12th week of life.

This report presents changes of IGFs and IGFBPs in a female infant with partial trisomy 9q in the 12th week of life. Studying deficient growth in this hypoplastic infant (birth weight 1405 g, birth length 36 cm) with dysmorphic features, the following changes in IGFs and IGFBPs were detected (microg/l): IGF-I: 26.5 vs 48.1 in healthy infants; IGF-II: 420 vs 728; IGFBP-2: 931 vs 524; IGFBP-3: 800 vs 1070. This demonstrates that IGFs and IGFBPs may reflect individual insufficient growth even at this early age.

Klinik, Radiologie, Genetik und Therapie hereditärer Skeletterkrankungen Ein historischer Überblick anläßlich des 100. Geburtstages von Professor Erich Häßler

In diesem Beitrag werden klinische Symptomatik, Radiologie, Genetik und die Therapie spezieller Skeletterkrankungen, soweit möglich unter Bezugnahme auf eigene Patienten, diskutiert. Ausgewählt sind hier die Mucopolysaccharidose Typ I-S (Scheie-Syndrom), das Okzipitalhorn-Syndrom und die autosomal-rezessiv erbliche Osteopetrose. Die Arbeit ist Herrn Professor Erich Häßler gewidmet, der 1999 seinen 100.Geburtstag beging. Professor Häßler war von 1953 bis 1965 Direktor der Jenaer Kinderklinik und beschäftigte sich wissenschaftlich mit verschiedenen Knochenerkrankungen wie der Osteopetrose, der Chondrodysplasia punctata und der Mucopolysaccharidose I-H (Hurler- Syndrom).