Carl Birger van der Hagen

Direct Diagnosis by DNA Analysis of the Fragile X Syndrome of Mental Retardation

Abstract Background. The fragile X syndrome, the most common form of inherited mental retardation, is caused by mutations that increase the size of a specific DNA fragment of the X chromosome (in Xq27.3). Affected persons have both a full mutation and abnormal DNA methylation. Persons with a smaller increase in the size of this DNA fragment (a premutation) have little or no risk of retardation but are at high risk of having affected children or grandchildren. The passage from premutation to full-mutation status occurs only with transmission from the mother. We have devised a method of identifying carriers of these mutations by direct DNA analysis. Method. We studied 511 persons from 63 families with the fragile X syndrome. Mutations and abnormal methylation were detected by Southern blotting with a probe adjacent to the mutation target. Analysis of EcoRI and EagI digests of DNA distinguished clearly in a single test between the normal genotype, the premutation, and the full mutation. Results. DNA analysis...

An excess of chromosome 1 breakpoints in male infertility.

In a search for potential infertility loci, which might be revealed by clustering of chromosomal breakpoints, we compiled 464 infertile males with a balanced rearrangement from Mendelian Cytogenetics Network database (MCNdb) and compared their karyotypes with those of a Danish nation-wide cohort. We excluded Robertsonian translocations, rearrangements involving sex chromosomes and common variants. We identified 10 autosomal bands, five of which were on chromosome 1, with a large excess of breakpoints in the infertility group. Some of these could potentially harbour a male-specific infertility locus. However, a general excess of breakpoints almost everywhere on chromosome 1 was observed among the infertile males: 26.5 versus 14.5% in the cohort. This excess was observed both for translocation and inversion carriers, especially pericentric inversions, both for published and unpublished cases, and was significantly associated with azoospermia. The largest number of breakpoints was reported in 1q21; FISH mapping of four of these breakpoints revealed that they did not involve the same region at the molecular level. We suggest that chromosome 1 harbours a critical domain whose integrity is essential for male fertility.

Congenital dislocation of the hip joint in Norway V. Evaluation of genetic and environmental factors

The incidence of congenital dislocation of the hip joint (CDH) in Norway is high. Environmental factors and familial occurrence of CDH have been studied in 1147 probands with neonatal CDH and in 784 probands with late‐diagnosis CDH. The proportion of affected sibs was 6 per cent in neonatal CDH and 8.5 per cent in late‐diagnosis CDH. In 51 families, patients with neonatal CDH had sibs with late‐diagnosis CDH, most of whom had been screened for CDH in the neonatal period. The distribution in families was computable with a polygenic mode of inheritance, and the heritability of CDH was calculated to be 74 per cent.

X‐linked aqueductal stenosis

A family is reported in which eight members of one generation were affected by the syndrome hydrocephalus with aqueductal stenosis. With the exception of one child who lived for several weeks, they all died at or within 10 days of birth. Autopsy of a pair of affected twins showed marked stenosis of the aqueduct of Sylvius with fusion of the lamina quadrigemina. There were no signs of previous or present inflammatory changes or neo‐plasia. All the affected individuals were males, and the familial and pathological data presented support the concept that aqueductal stenosis in this family was due to an X‐linked gene, and may have a developmental origin.

Polycythemia Vera treated with 32P and Myleran: Development of chronic granulocytic leukemia with chromosomal abnormalities in one patient

Chronic granulocytic leukemia developed in a 59–year‐old woman who had previously received a total of 21 mCi 32P for polycythemia Vera. She was treated with Myleran (busulphan) for her chronic granulocytic leukemia. Cytogenetic studies revealed deletion of chromosomes No. 8 and 12, and translocation between 1 and 8. The patient also developed a severe autoimmune hemolytic anemia, for which she received prednisone treatment. She died with a perforated stomach ulcer.

Studies of human chromosomes by DNA‐binding fluorochromes I. The normal chromosome complement

Casperssons method of labelling chromosomes with DNA‐binding fluorescent agents has been applied to the study of human chromosomes. Fluorescence distribution curves of normal metaphase chromosomes treated with quinacrine mustard (QM) were obtained by scanning transparent pictures of the labelled chromosomes in a Beckman Analytrol, an instrument normally used for scanning electrophoresis strips. Representative fluorescence distribution curves of the different chromosomes, as well as one complete “QM karyotype”, have been presented. The distribution curves of individual chromosomes appear to be characteristic and reproducible and it was concluded that the technique of fluorescent labelling holds great promise for identification of individual human chromosomes and chromosomal regions.

A de novo 6p interstitial deletion and a complex translocation involving chromosomes 2, 6, and 14 in a mildly developmentally delayed patient.

Constitutional complex chromosome rearrangements (CCRs) involve two or more breakpoints with exchange of segments among at least two chromosomes [Pai et al., 1980]. A combined technical approach (G banding, aCGH, and FISH) documented a de novo CCR in our patient. The patient was born at term with normal birth weight after an uneventful pregnancy. He has healthy, nonconsanguineous parents (the mother was 35, the father 38 years old), and healthy siblings. As a baby he was quiet and slept excessively. He walked at 15 months. At 3 years he was referred for a chromosome analysis because of global delayed development and minor anomalies. At 5 years he lagged around 1 year behind his peers. Language development was most delayed, especially pronunciation. At 5 years he could put two to three words together, but it was difficult to understand him. No anatomic malformations were identified that could explain his language difficulties. Motor development was only slightly delayed and no hypotonia was noted. He also has deep set eyes, a prominent philtrum, and a slightly prominent forehead (Fig. 1). In addition, his ears are slightly low set and posteriorly angulated, with normal shape. A physical eye examination at the age of 6.5 years old showed: (1) 3 mm chorioretinal coloboma inferonasally in the left eye; (2) hypermetropia of 2.5 diopters bilaterally and minor exophoria; (3) hypertelorism with an interpupillary distance (IPD) of 62 mm (>97th centile), inner canthal distance (ICD) of 35 mm (97th centile), and outer canthal distance (OCD) of 84 mm (75th centile) [Dollfus and Verloes, 2004]. He has atopic dermatitis and asthma. Gbanding at standard resolution, followedbyFISH with Whole Painting Libraries from chromosomes 2, 6, and 14, and with 2qtel and 6qtel probes (Vysis, Abbott Molecular Inc., Downers Grove, IL), defined the karyotype of the propositus as: 46,XY, t(2,6,14) der (2)(2pter ! 2q33.3::6p24.1 ! 6pter), der(6) (2qter>2q33.3::14q24.3! 14q11.2::6p24.1! 6qter), der(14)(14pter ! 14q11.2::14q24.3 ! 14qter).ish der(2)(wcp2þ,wcp6þ,tel6pþ).ish der(6)(wcp6þ, wcp14þ ,wcp2þ , tel2qþ ) . ish der (14)(wcp14þ) (Fig. 2). Array-CGH (44k B oligo array, Agilent Technologies, Santa Clara, CA; data analysis by BlueFuse, BlueGnome, Cambridge, UK) detected a deletion on chr6:7933252–10510600 bp, a duplication on chr7:142863310–143726151 bp, and a deletion on chr14:18590766–19851375 bp (supporting information Table I may be found in the online version of this article). Thepositionsof the aberrations refer toNCBI Build 35. The duplicated region on chromosome 7 was not studied further because it corresponds to a known Copy Number Variant (CNV, http://projects.tcag.variation). The chromosome 14 deletion also corresponds to a known CNV and it is enriched with intraand inter-chromosome segmental duplications. This region, however, was analyzed by FISH

Studies of human chromosomes by DNA-binding fluorochromes: II. Fluorescence characteristics of the supernumerary G chromosome in Down's syndrome

Previous work suggested that chromosomes 21 and 22 could be distinguished by fluorescent labelling of the chromosomes with quinacrine mustard (QM). This suggestion was confirmed in the present study of patients with Downs syndrome since it was found that in most cases three of the five G chromosomes had a clearly different distribution pattern from the remaining two. The pattern typical of chromosome 21 displays a particularly bright fluorescence in the middle of the long arm, whereas the fluorescence of chromosome 22 is less intense and more equally distributed along the length of the chromosome. Individual patterns of fluorescence have been observed in chromosome 21. Some of the implications of the findings are discussed.

A partial trisomy 1q patient with a deletion 1q22 and an insertion 1(q42q44) into 1q22

We report on a patient whose karyotype was defined to be 46,XY,der(1).ish del (q22) ins (q22q44q42) dup (q42q44), by combining G banding, aCGH, and FISH studies. As a child the patient had distinctive features, with a broad flattened nasal bridge, slight hypertelorism, downward slanted eyes, bushy eyebrows, divergent strabismus, trigonocephaly with a prominent forehead, short stubby nose, triangular mouth, high palate, long prominent philtrum and hypognathia (Fig. 1a). Birth weight is not known. He started to walk late and speech was delayed and difficult to understand. As a grown-up he has a long narrow face, is friendly and pleasant with a slurred pronunciation, functioning at about IQ 50–60. He has low hemoglobin with high transferrin values, but no known heart defects. Otherwise he is healthy with normal stature. He is now 53 years old, he lives in an institution and he is sociable. G-banding detected an abnormal chromosome 1 (Fig. 1b), which apparently contained a duplicated region in 1q24. aCGH (44K oligo array, Agilent Technologies, Santa Clara, CA; data analysis by BlueFuse, BlueGnome, Cambridge, UK) detected three aberrations: (1) 1p36.13 deletion (chr1:16686959–16794038 bp), (2) 1q22 deletion (chr1:152396245–152647439 bp), (3) 1q42–q44 duplication (chr1:221209415–245422360 bp) (supporting information Table I may be found in the online version of this article). The positions of the aberrations refer to the NCBI Build 35. The deletion in 1p36.13 is a well known copy number variation, detected in several independent studies (http://projects.tcag.variation), and it does not overlap with the region previously reported to be involved in the 1p36 microdeletion syndrome [Heilstedt et al., 2003]. This deletion was not verified by FISH because we assume that it does not contribute to the pathological phenotype in our patient. The 250 kb deletion in chromosome 1q22 was confirmed by FISH using the BAC clones RP11-586I23 and RP11-243J18 which gave signal on chromosome 1, but not on der(1) (Fig. 2a,b and Table I). This region includes six genes: one gene encoding a hypothetical protein in addition to the genes; ASH1L, MSTO1, YY1AP1, DAP3, and GON4L. Because of the limited information about the function of these genes, we are not able to hypothesize the contribution of this deletion to the clinical phenotype of the patient. The third aberration detected by aCGH is a 23 Mb duplication. Even though the G-banding result indicated a duplication in chromosome 1q24, the duplication detected by aCGH maps in chromosome 1q42.1! q44. FISH experiments revealed that (1) the duplicated segment (from RP11-55A11 to RP11-103M14, see Table I) is inserted in 1q in a proximal region and (2) it has inverted orientation compared to its original location (Fig. 2c). We then hypothesized that the duplicated 1q42.1! q44-segment is inserted in 1q22, in connection with the deletion. The FISH results confirmed this hypothesis, since the two clones flanking the deletion (RP11-313J15 and RP11-1141E12) are located at the borders of the duplicated region in this patient (Fig. 2d,e). No close relatives are available for investigations, and there is no family history suggesting the presence of inherited chromosome abnormalities. The 1q22 deletion has a nearly perfect overlap with the SD1q22 region [Kuryshev et al., 2006], consisting of two segmental duplications head-tail oriented, spanning about 240 kb originated from an Alu-Alu mediated recombination about 37 million of years ago. The region contains approximately 40% Alu sequences, which is an enrichment compared to the genome average of 10.6% [Human genome consortium, 2001]. The abundance of Alu elements could facilitate the double crossover event resulting in the der(1) detected