Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.

Interstitial deletions of the chromosome 22q11.2 region are the most common microdeletions in humans. The TBX1 gene is considered to be the major candidate gene for the main features in 22q11.2 deletion syndrome, including congenital heart malformations, (para)thyroid hypoplasia, and craniofacial abnormalities. We report on eight patients with atypical deletions of chromosome 22q11.2. These deletions comprise the distal part of the common 22q11.2 deleted region but do not encompass the TBX1 gene. Ten similar patients with overlapping distal 22q11.2 deletions have been reported previously. The clinical features of these patients are described and compared to those found in the classic 22q11.2 deletion syndrome. We discuss the possible roles of a position effect or haploinsufficiency of distally located genes (e.g., CRKL) in the molecular pathogenesis of the 22q11.2 deletion syndrome.

Cat eye syndrome (CES) is caused by a gain of the proximal part of chromosome 22. Usually, a supernumerary marker chromosome is present, containing two extra copies of the chromosome 22q11.1q11.21 region. More sporadically, the gain is present intrachromosomally. The critical region for CES is currently estimated to be about 2.1 Mb and to contain at least 14 RefSeq genes. Gain of this region may cause ocular coloboma, preauricular, anorectal, urogenital and congenital heart malformations. We describe a family in which a 600 kb intrachromosomal triplication is present in at least three generations. The copy number alteration was detected using MLPA and further characterized with interphase and metaphase FISH and SNP-array. The amplified fragment is located in the distal part of the CES region. The family members show anal atresia and preauricular tags or pits, matching part of the phenotype of this syndrome. This finding suggests that amplification of the genes CECR2, SLC25A18 and ATP6V1E1, mapping within the critical region for CES, may be responsible for anorectal, renal and preauricular anomalies in patients with CES.

We report on a 13-year-old female patient, born from healthy, non-consanguineous parents. She was born at 37 weeks gestation by cesarean section because of meconium stained amniotic fluid and late fetal heart decelerations. After birth she required assisted ventilation because of respiratory insufficiency. During intubation a posterior mucosal cleft palate and bifid uvula were noted. She was small for gestational age (weight 1,780 gram, −3 SD; head circumference at −1.2 SD) and showed dysmorphic features with frontal bossing, rethrognatia, hyperplastic gums, large fontanelles, single transverse palmar creases, ulnar deviation of wrists, bilateral clubfeet and syndactyly of toes 1-5th. No cardiac or other visceral abnormalities were detected.In time, developmental milestones were delayed. Her growth rate was also delayed and at the age of 6 years ( Figure 1a) height followed the −4 SD, with an average head circumference for the age. Examination at age 6 years showed metopic synostosis, high-arched eyebrows, flattened orbital ridges, small chin, prominent front teeth, short philtrum, congenital contractures of fingers, elbows and knees (Figure 1b), broad thorax with widely separated nipples (Figure 1c), thoracic scoliosis, high-pitched voice, nasal speech and hypotonia. Failure to thrive required gastric tube feedings. She also presented with mild-moderate intellectual disability. Brain MRI at 4 years showed a retrocerebellar arachnoidal cyst and thin corpus callosum. Metabolic screening shortly after birth was normal. Because she displayed mild features resembling Bohring-Opitz trigonocephaly, the ASXL1 gene was analyzed by capillary sequencing of the coding regions in DNA extracted from blood. No pathogenic variants in this gene were detected. G-banded chromosome analysis shortly after birth revealed a 45,X complement in peripheral blood, consistent with Turner syndrome. As the girl's phenotype could not be explained by this Turner karyotype, additional genetic analyses were performed. Subtelomeric MLPA (SALSA P036C en P070 kits, MRC Holland) and genomic array (Affymetrix 250 k Nsp1 array) analysis using DNA obtained from peripheral blood confirmed the 45,X karyotype.Additionally, array showed a small variant of unknown significance, inherited from the healthy father (114 kb interstitial loss on chromosome 4q31.22, containing part of the glycophorin E and B genes).As still no satisfactory explanation for the girl's phenotype was found, a mosaic genetic aberration was suspected. Therefore, genomic array was repeated on the Illumina HumanCyto12-SNP array using DNA obtained from a skin biopsy. An abnormal profile was observed, suggesting the presence of a triploid cell line with loss of one X chromosome. Based on the B-allele frequency, this cell line could possibly be present in a high mosaic form, that is, in more than 75% of the cells (Figure 2a). Subsequent karyotyping on cultured skin fibroblasts indeed showed a mosaic karyotype: 68,XX[5]/45,X[1] (Figure 2b), confirming the array findings.In conclusion, the pat...

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For suppression of primary tumor growth and metastatic spread, aspirin and theophylline, either alone or combined, were given daily to inbred female BN rats after sc implantation of a syngeneic nonimmunogenic tumor. Treatment with 200 mg aspirin/kg (body wt) resulted in a statistically significant regression of tumor growth as well as of the number of metastases in the lungs. Aspirin given in a lower dose (20 mg/kg) did not show significant difference from the vehicle group. Theophylline (75 mg/kg) significantly increased primary tumor growth as well as lung metastases. Inhibition of in vitro platelet aggregation, determined in whole blood taken from non-tumor-bearing animals treated with the same therapeutic regimen, was most pronounced in those groups in which tumor growth and spread were significantly retarded. However, this positive correlation between inhibition of tumor spread and platelet aggregation was not associated with a favorable balance of prostacyclin and thromboxane A2 in these animals.

Diets high (17.7 cal%) and low (3.3 cal%) in linoleic acid were given to groups of Brown Norway female rats before and after inoculation of syngeneic tumor models with different characteristics, with regard to tumor spread, malignancy, immunogenicity, growth rate, rat strain, and histopathological features. Despite the differences in characteristics, in most tumor models, tumor growth was identical in both experimental groups. However, in 2 tumor models, an adrenal cortical carcinoma and a myeloid leukemia, differences in growth were noted. In rats given the diet low in linoleic acid, growth of the cortical carcinoma was significantly increased, whereas the opposite effect was seen in rats with myeloid leukemia.