Gotthold Barbi

Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features

Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.

Molecular cytogenetic analysis of a familial interstitial deletion Xp22.2-22.3 with a highly variable phenotype in female carriers.

We describe a familial interstitial deletion of 7.7‐Mb involving Xp22.2‐22.3. The deletion was transmitted from an asymptomatic mother to her two children with severe developmental delay, no speech development and autistic behavior. Assessment of the deletion boundaries by FISH and PCR analyses indicated that the deletions encompasses 27 genes. Several of these genes are associated with known disorders, like KAL1 (Kallmann syndrome), steroid sulfatase (STS) (X‐linked ichtyosis), and arylsulfatase E (ARSE) (chondrodysplasia punctata). The deletion also includes all four VCX genes (VCX‐A, VCX‐B1, VCX‐B, and VCX‐C) and the neuroligin 4 (NLGN4) gene. VCX‐A deficiency has been shown previously to be associated with mental retardation and NLGN4 mutations lead to mental retardation in conjunction with autism. Functional deficiency of both MRX genes, VCX‐A and NLGN4, are most likely associated with the impaired cognitive development of the patients described here. The phenotype associated with the Xp deletion was highly variable in female carriers and might be attributed to unfavorable X inactivation. However, all the 27 genes included in the deleted interval escape X inactivation and are expressed at variable levels from the normal X chromosome. Thus, the overall X inactivation pattern and inter‐individual expression variability of the genes in distal Xp might be determinants of the phenotype associated with the deletion.

Disruptions of the novel KIAA1202 gene are associated with X-linked mental retardation

The extensive heterogeneity underlying the genetic component of mental retardation (MR) is the main cause for our limited understanding of the aetiology of this highly prevalent condition. Hence we set out to identify genes involved in MR. We investigated the breakpoints of two balanced X;autosome translocations in two unrelated female patients with mild/moderate MR and found that the Xp11.2 breakpoints disrupt the novel human KIAA1202 (hKIAA1202) gene in both cases. We also identified a missense exchange in this gene, segregating with the Stocco dos Santos XLMR syndrome in a large four-generation pedigree but absent in >1,000 control X-chromosomes. Among other phenotypic characteristics, the affected males in this family present with severe MR, delayed or no speech, seizures and hyperactivity. Molecular studies of hKIAA1202 determined its genomic organisation, its expression throughout the brain and the regulation of expression of its mouse homologue during development. Transient expression of the wild-type KIAA1202 protein in HeLa cells showed partial colocalisation with the F-actin based cytoskeleton. On the basis of its domain structure, we argue that hKIAA1202 is a new member of the APX/Shroom protein family. Members of this family contain a PDZ and two ASD domains of unknown function and have been shown to localise at the cytoskeleton, and play a role in neurulation, cellular architecture, actin remodelling and ion channel function. Our results suggest that hKIAA1202 may be important in cognitive function and/or development.

Nonrandom distribution of methotrexate-induced aberrations on human chromosomes. Detection of further folic acid sensitive fragile sites

SummaryEleven folic acid sensitive fragile sites (3p14, 7p13, 7q31.1, 7q32, 9q32, 11p13, 14q23, 15q22, 16q23, Xp22.2, Xq22) were detected in one individual, eight of them previously unknown. These sites seem to bear each its specific sensitivity to folic acid deficiency. Six of the sites were observed simultaneously on both homologous chromosomes in at least one cell. Each of these 11 sites was also found in at least one among 12 individuals further examined. Some of these individuals showed six of these 11 sites. The fragile site 3p14 was detected in all individuals examined. The homologous sites 3p14 of one individual differed from each other in their frequency of lesions induced by methotrexate as well as fluorodeoxyuridine. This observation suggests that folic acid sensitivity is a property inherent in the chromatin of an individual chromosome at the site involved in fragility. This property seems to be responsible for the nonrandom fragility at that site and also for the individual sensitivity of each chromosomal site.

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.

Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells.

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.

Expression of the fragile site Xq27 in fibroblasts. I. Detection of fra(X)(q27) in fibroblast clones from males with X-linked mental retardation.

SummaryTwelve fibroblast clones from two males with X-linked mental retardation expressed the fragile site Xq27 in 3%–38% of metaphases analyzed. The number of in vitro doublings during the cloning procedure had no evident influence on the induction of fragile X expression. The variability of fragile X expression seems to depend on cell properties acquired during culture rather than on properties originally inherent in the cells.

Characterization of a 16 Mb interstitial chromosome 7q21 deletion by tiling path array CGH

We report on a 42‐year‐old female patient with an interstitial 16 Mb deletion in 7q21.1‐21.3 and a balanced reciprocal translocation between chromosomes 6 and 7 [karyotype 46,XX,t(6;7)(q23.3;q32.3)del(7)(q21.1q21.3)de novo]. We characterized the size and position of the deletion by tiling path array comparative genomic hybridization (CGH), and we mapped the translocation breakpoints on chromosomes 6 and 7 by FISH. The clinical features of this patient—severe mental retardation, short stature, microcephaly and deafness—are in accordance with previously reported patients with 7q21 deletions. Chromosome band 7q21.3 harbors a locus for split hand/split foot malformation (SHFM1), and part of this locus, including the SHFM1 candidate genes SHFM1, DLX5, and DLX6, is deleted. The absence of limb abnormalities in this patient suggests either a location of the SHFM1 causing factor distal to this deletion, or reduced penetrance of haploinsufficiency of a SHFM1 factor within the deleted interval.

ICF syndrome: high variability of the chromosomal phenotype and association with classical Hodgkin lymphoma.

We report on two sibs with ICF syndrome (immunodeficiency, centromeric heterochromatin instability, and facial anomalies) diagnosed in the elder brother based on the typical chromosomal abnormalities present in 56% of metaphases from cultured lymphocytes. In a previous cytogenetic analysis this diagnosis had been missed due to low manifestation of the ICF chromosomal phenotype. Hypomethylation of classical satellites 2 and 3, and of α‐satellite DNA was shown in the lymphocytes of the younger sister. At 7 years of age the boy presented with hemiplegia due to tumerous invasion of the right brachial plexus. Histopathology revealed classical Hodgkin lymphoma, a neoplasia which might have been facilitated by the underlying genetic defect.