G.J.B. van Ommen

Transcriptome and genome sequencing uncovers functional variation in humans.

Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of messenger RNA and microRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project—the first uniformly processed high-throughput RNA-sequencing data from multiple human populations with high-quality genome sequences. We discover extremely widespread genetic variation affecting the regulation of most genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on the cellular mechanisms of regulatory and loss-of-function variation, and allows us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.

PRENATAL-DIAGNOSIS AND CARRIER DETECTION OF DUCHENNE MUSCULAR-DYSTROPHY WITH CLOSELY LINKED RFLPS

By the use of a series of closely linked DNA probes detecting restriction fragment length polymorphisms (RFLPs) distributed over the short arm of the X chromosome, a double crossover was detected in a Duchenne muscular dystrophy carrier and an affected male fetus was diagnosed at 12 weeks of gestation, with a probable accuracy of more than 99.0%. A new mutation was identified in another family with the same degree of reliability; three females in this family were thus deemed not to be DMD carriers. The eleven RFLP-markers presently available on the short arm of the X chromosome are useful in the diagnosis of DMD since they bridge the Duchenne locus at genetic distances varying between 3 and 20 cmo. Moreover, recombination within the set of markers provides an independent way of regionally mapping these probes relative to each other along the short arm of the X chromosome.

Duchenne muscular dystrophy High frequency of deletions

DNA probes are available for Duchenne muscular dystrophy (DMD) carrier detection and prenatal diagnosis. With probes for about 25% of the proximal portion of the gene, we found the proximal probes detected deletions in 23% of nonselected DMD boys, while a single distal probe detected 17% more as deletions. The combined percentage was 39% for all probes tested. Prenatal diagnosis and carrier detection are more accurate if deletions are mapped rather than by use of restriction fragment length polymorphism analysis. The effort involved in screening all affected boys for deletions is considerably less, and provides an accurate genetic marker for subsequent prenatal diagnosis in the family and prospective counseling for female relatives. It seems likely that, once the entire gene (cDNA) is available for screening, most DMD boys will show deletions.

Rapid subchromosomal localization of cosmids by nonradioactive in situ hybridization

A rapid method for localizing large numbers of complete cosmids by nonradioactive in situ hybridization is described. The cosmids are nick translated in the presence of biotin-16-dUTP, incubated with an excess of sonicated human DNA, and used as a probe for in situ hybridization. Sites of hybridization are detected by successive treatments with FITC-labeled avidin and biotinylated anti-avidin antibody. Fifty-two cosmids were localized on chromosome 16 in 5 d relative to translocation breakpoints contained in two cell lines. Rapid identification of chromosome 16 was achieved by cohybridization with a chromosome 16-specific centromeric repeat probe.

Isolation of probes detecting restriction fragment length polymorphisms from X chromosome-specific libraries: potential use for diagnosis of Duchenne muscular dystrophy

SummaryWe have isolated 23 human X chromosome-specific DNA fragments from λ libraries, prepared from flow-sorted X chromosomes. To increase diagnostic potential for X-linked genetic disorders, including Duchenne muscular dystrophy (DMD), the fragments were tested for restriction fragment length polymorphisms (RFLPs) with six restriction enzymes. All fragments were regionally mapped to segments of the X chromosome with a panel of somatic cell hybrids and with human cell lines carrying unbalanced chromosomal abnormalities. Two of the isolated probes detected a high frequency RFLP. One, 754, maps between Xp11.3 and Xp21 and detects a PstI polymorphism with an allele frequency of 0.38. The other, 782, maps between Xp22.2 and Xp22.3 and reveals an EcoRI polymorphism with an allele frequency of 0.40. According to a pilot linkage study of families at risk for Duchenne muscular dystrophy, 754 gives a maximum Lod score of 7.6 at a recombination fraction of 0.03. Probe 782 lies telomeric to DMD with a maximum Lod score of 2.2 at a recombination fraction of 0.17. Using our X-chromosomal probes and a set of autosomal probes, isolated and examined in an identical way, we found a significantly lower RFLP frequency for the X chromosome as compared to the autosomes.

Germinal mosaicism increases the recurrence risk for 'new' Duchenne muscular dystrophy mutations.

In 288 Dutch and Belgian Duchenne and Becker muscular dystrophy families, the parental origin of 41 new deletion or duplication mutations was determined. Twenty seven of the new mutations occurred in the maternal X chromosome and nine in the grandmaternal and five in the grandpaternal X chromosome. The grandparental data are compatible with equal mutation rates for DMD in male and female X chromosomes. New mutations were defined by their presence in one or more progeny and absence in the lymphocytes of the mother or the grandparents. In one family a fraction of the maternal lymphocytes was found to carry the mutation, suggesting somatic mosaicism. In six cases out of 41, the mutation was transmitted more than once by a parent in whom the mutation was absent in lymphocytes, suggesting gonadal mosaicism as the explanation for the multiple transmission. Using our data for the recurrence of the mutations among the total of at risk haplotypes transmitted, we arrive at a recurrence risk of 14% for the at risk haplotype. The observation of this high risk of germinal mosaicism is crucially important for all physicians counselling females in DMD families. Recently, germinal mosaicism has been observed also in a number of other X linked and autosomal disorders. The implications and appropriate diagnostic precautions are discussed.

A physical map of 4 million bp around the Duchenne muscular dystrophy gene on the human X-chromosome

Employing pulsed field gradient electrophoresis, we constructed a 4.5 million bp (Mb) Sfil restriction map of the human X-chromosomal region p21, harboring genes for Duchenne (DMD) and Becker Muscular Dystrophy. In a DMD patient with additional chronic granulomatosis and retinitis pigmentosa, the proximal 3.5 Mb is deleted. Another DMD patient, with additional glycerol kinase deficiency and adrenal hypoplasia, lacks at least 3.3 Mb in the middle region, including marker C7 but not B24, placing C7 closer to DMD. Another DMD patient has a partial pERT-87 deletion of minimally 140 kb. Truncated Sfil fragments in a female X:21 translocation patient place the junction probe XJ1.1 115 kb from the distal end of the normal fragment. Probe pERT-84 maps to the same fragment, within 750 kb of XJ1.1.

Serum matrix metalloproteinase-9 (MMP-9) as a biomarker for monitoring disease progression in Duchenne muscular dystrophy (DMD)

To identify serum biomarkers that allow monitoring of disease progression and treatment effects in Duchenne muscular dystrophy (DMD) patients, levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1) and osteopontin (OPN) were determined in 63 DMD patients on corticosteroid therapy. These proteins were selected for their role in the pathogenesis of muscular dystrophy. Levels of MMP-9 and TIMP-1 were significantly higher in sera of DMD patients compared to healthy controls, whereas the OPN levels showed no significant difference. MMP-9 levels were also observed to be significantly higher in older, nonambulant patients, compared to ambulant patients. Longitudinal data from a smaller cohort of DMD patients followed up for over 4years showed that MMP-9, but not TIMP-1 increased significantly with age. Hence, MMP-9 is a potential DMD biomarker for disease progression. Future studies have to confirm whether serum MMP-9 levels can be used to monitor therapeutic response.

Diagnostic analysis of the Rubinstein-Taybi syndrome: five cosmids should be used for microdeletion detection and low number of protein truncating mutations

Rubinstein-Taybi syndrome (RTS) is a malformation syndrome characterised by facial abnormalities, broad thumbs, broad big toes, and mental retardation. In a subset of RTS patients, microdeletions, translocations, and inversions involving chromosome band 16p13.3 can be detected. We have previously shown that disruption of the human CREB binding protein (CREBBP orCBP) gene, either by these gross chromosomal rearrangements or by point mutations, leads to RTS. CBP is a large nuclear protein involved in transcription regulation, chromatin remodelling, and the integration of several different signal transduction pathways. Here we report diagnostic analysis of CBP in 194 RTS patients, divided into several subsets. In one case the mother is also suspect of having RTS. Analyses of the entireCBP gene by the protein truncation test showed 4/37 truncating mutations. Two point mutations, one 11 bp deletion, and one mutation affecting the splicing of the second exon were detected by subsequent sequencing. Screening theCBP gene for larger deletions, by using different cosmid probes in FISH, showed 14/171 microdeletions. Using five cosmid probes that contain the entire gene, we found 8/89 microdeletions of which 4/8 were 5′ or interstitial. This last subset of microdeletions would not have been detected using the commonly used 3′ probe RT1, showing the necessity of using all five probes.

Duration of illness in Huntington's disease is not related to age at onset.

The age at onset and duration of illness were studied in patients with Huntingtons disease in the Leiden Roster which at 1 July 1990 contained 2787 patients. Of 1106 patients, 800 deceased and 306 alive, the age at onset was known. The median duration was 16.2 (range 2-45) years. In contrast to the current opinion, the median duration was independent of the age of onset. The median duration in juvenile Huntingtons disease was 17.1 years, which is much longer than reported in the literature, and comparable with the categories for the age of onset of 20-34 and 35-49 years. Only in the group where onset was over 50 years of age was the median duration somewhat shorter (15.6 years), which can be ascribed to unrelated causes of death. As age of onset and duration of illness are not related, at least two mechanisms to determine the clinical course have to be postulated: one for age of onset and another for duration of illness. Duration was shorter for males, especially for those with an affected father.