Ludovica Volpi

Rothmund-Thomson syndrome

Rothmund-Thomson syndrome (RTS) is a genodermatosis presenting with a characteristic facial rash (poikiloderma) associated with short stature, sparse scalp hair, sparse or absent eyelashes and/or eyebrows, juvenile cataracts, skeletal abnormalities, radial ray defects, premature aging and a predisposition to cancer. The prevalence is unknown but around 300 cases have been reported in the literature so far. The diagnostic hallmark is facial erythema, which spreads to the extremities but spares the trunk, and which manifests itself within the first year and then develops into poikiloderma. Two clinical subforms of RTS have been defined: RTSI characterised by poikiloderma, ectodermal dysplasia and juvenile cataracts, and RTSII characterised by poikiloderma, congenital bone defects and an increased risk of osteosarcoma in childhood and skin cancer later in life. The skeletal abnormalities may be overt (frontal bossing, saddle nose and congenital radial ray defects), and/or subtle (visible only by radiographic analysis). Gastrointestinal, respiratory and haematological signs have been reported in a few patients. RTS is transmitted in an autosomal recessive manner and is genetically heterogeneous: RTSII is caused by homozygous or compound heterozygous mutations in the RECQL4 helicase gene (detected in 60-65% of RTS patients), whereas the aetiology in RTSI remains unknown. Diagnosis is based on clinical findings (primarily on the age of onset, spreading and appearance of the poikiloderma) and molecular analysis for RECQL4 mutations. Missense mutations are rare, while frameshift, nonsense mutations and splice-site mutations prevail. A fully informative test requires transcript analysis not to overlook intronic deletions causing missplicing. The diagnosis of RTS should be considered in all patients with osteosarcoma, particularly if associated with skin changes. The differential diagnosis should include other causes of childhood poikiloderma (including dyskeratosis congenita, Kindler syndrome and Poikiloderma with Neutropaenia), other rare genodermatoses with prominent telangiectasias (including Bloom syndrome, Werner syndrome and Ataxia-telangiectasia) and the allelic disorders, RAPADILINO syndrome and Baller-Gerold syndrome, which also share some clinical features. A few mutations recur in all three RECQL4 diseases. Genetic counselling should be provided for RTS patients and their families, together with a recommendation for cancer surveillance for all patients with RTSII. Patients should be managed by a multidisciplinary team and offered long term follow-up. Treatment includes the use of pulsed dye laser photocoagulation to improve the telangiectatic component of the rash, surgical removal of the cataracts and standard treatment for individuals who develop cancer. Although some clinical signs suggest precocious aging, life expectancy is not impaired in RTS patients if they do not develop cancer. Outcomes in patients with osteosarcoma are similar in RTS and non-RTS patients, with a five-year survival rate of 60-70%. The sensitivity of RTS cells to genotoxic agents exploiting cells with a known RECQL4 status is being elucidated and is aimed at optimizing the chemotherapeutic regimen for osteosarcoma.

Targeted next-generation sequencing appoints c16orf57 as clericuzio-type poikiloderma with neutropenia gene.

Next-generation sequencing is a straightforward tool for the identification of disease genes in extended genomic regions. Autozygosity mapping was performed on a five-generation inbred Italian family with three siblings affected with Clericuzio-type poikiloderma with neutropenia (PN [MIM %604173]), a rare autosomal-recessive genodermatosis characterised by poikiloderma, pachyonychia, and chronic neutropenia. The siblings were initially diagnosed as affected with Rothmund-Thomson syndrome (RTS [MIM #268400]), with which PN shows phenotypic overlap. Linkage analysis on all living subjects of the family identified a large 16q region inherited identically by descent (IBD) in all affected family members. Deep sequencing of this 3.4 Mb region previously enriched with array capture revealed a homozygous c.504-2 A>C mismatch in all affected siblings. The mutation destroys the invariant AG acceptor site of intron 4 of the evolutionarily conserved C16orf57 gene. Two distinct deleterious mutations (c.502A>G and c.666_676+1del12) identified in an unrelated PN patient confirmed that the C16orf57 gene is responsible for PN. The function of the predicted C16orf57 gene is unknown, but its product has been shown to be interconnected to RECQL4 protein via SMAD4 proteins. The unravelled clinical and genetic identity of PN allows patients to undergo genetic testing and follow-up.

Chromosomal instability in fibroblasts and mesenchymal tumors from 2 sibs with Rothmund-Thomson syndrome.

Rothmund‐Thomson syndrome (RTS) is a rare autosomal recessive genodermatosis associated with increased risk of mesenchymal tumors. The putative gene has been provisionally assigned to chromosome 8. Using a cytogenetic‐molecular approach, we studied lymphocytes, fibroblasts, osteosarcoma (OS) and malignant fibrous histiocytoma (MFH) from 2 affected fraternal twins, looking for constitutive markers of chromosome instability and tumor chromosomal changes which might reflect the common genetic background. The rate of spontaneous chromosome aberrations was not increased in lymphocytes. Conversely, karyotyping of primary fibroblasts from one sib evidenced chromosome breaks and both numerical and structural chromosome changes in 24% and 17% of the metaphases respectively. FISH of a 8q21.3 cosmid allowed us to detect trisomy of the target region on 7% of fibroblast nuclei from both sibs, 47% and 12% of OS and MFH cells. Pronounced chromosomal instability and clonal rearrangements leading to different chromosome‐8 derivatives were detected in both tumors. CGH experiments showed multiple gains/losses, among which del(6q), also revealed by cytogenetics, and 7p gain were common, whereas 8q amplification was present only in OS. Chromosomal instability, observed in fibroblasts from the RTS patients studied, accounts for the increased risk of mesenchymal tumors in these patients. Int. J. Cancer 77:504–510, 1998.

Novel C16orf57 mutations in patients with Poikiloderma with Neutropenia: bioinformatic analysis of the protein and predicted effects of all reported mutations

BackgroundPoikiloderma with Neutropenia (PN) is a rare autosomal recessive genodermatosis caused by C16orf57 mutations. To date 17 mutations have been identified in 31 PN patients.ResultsWe characterize six PN patients expanding the clinical phenotype of the syndrome and the mutational repertoire of the gene. We detect the two novel C16orf57 mutations, c.232C>T and c.265+2T>G, as well as the already reported c.179delC, c.531delA and c.693+1G>T mutations. cDNA analysis evidences the presence of aberrant transcripts, and bioinformatic prediction of C16orf57 protein structure gauges the mutations effects on the folded protein chain.Computational analysis of the C16orf57 protein shows two conserved H-X-S/T-X tetrapeptide motifs marking the active site of a two-fold pseudosymmetric structure recalling the 2H phosphoesterase superfamily. Based on this model C16orf57 is likely a 2H-active site enzyme functioning in RNA processing, as a presumptive RNA ligase.According to bioinformatic prediction, all known C16orf57 mutations, including the novel mutations herein described, impair the protein structure by either removing one or both tetrapeptide motifs or by destroying the symmetry of the native folding.Finally, we analyse the geographical distribution of the recurrent mutations that depicts clusters featuring a founder effect.ConclusionsIn cohorts of patients clinically affected by genodermatoses with overlapping symptoms, the molecular screening of C16orf57 gene seems the proper way to address the correct diagnosis of PN, enabling the syndrome-specific oncosurveillance.The bioinformatic prediction of the C16orf57 protein structure denotes a very basic enzymatic function consistent with a housekeeping function. Detection of aberrant transcripts, also in cells from PN patients carrying early truncated mutations, suggests they might be translatable. Tissue-specific sensitivity to the lack of functionally correct protein accounts for the main cutaneous and haematological clinical signs of PN patients.

An unusual mutation in RECQ4 gene leading to Rothmund–Thomson syndrome

Rothmund-Thomson syndrome (OMIM #268400) is a severe autosomal recessive genodermatosis: characterised by growth retardation, hyperpigmentation and frequently accompanied by congenital bone defects, brittle hair and hypogonadism. Mutations in helicase RECQ4 gene are responsible for a subset of cases of RTS. Only six mutations have been reported, thus, far and each affecting the coding sequence or the splice junctions. We report the first homozygous mutation in RECQ4 helicase: 2746-2756-delTGGGCTGAGGC in IVS8 responsible for the severe phenotype associated with RTS in a Malaysian pedigree. We report also a 5321 G-->A transition in exon 17 and the updated list of the RECQ4 gene mutations.

Clericuzio-type poikiloderma with neutropenia syndrome in three sibs with mutations in the C16orf57 gene: delineation of the phenotype

We report on three sibs who have autosomal recessive Clericuzio‐type poikiloderma neutropenia (PN) syndrome. Recently, this consanguineous family was reported and shown to be informative in identifying the C16orf57 gene as the causative gene for this syndrome. Here we present the clinical data in detail. PN is a distinct and recognizable entity belonging to the group of poikiloderma syndromes among which Rothmund–Thomson is perhaps the best described and understood. PN is characterized by cutaneous poikiloderma, hyperkeratotic nails, generalized hyperkeratosis on palms and soles, neutropenia, short stature, and recurrent pulmonary infections. In order to delineate the phenotype of this rare genodermatosis, the clinical presentation together with the molecular investigations in our patients are reported and compared to those from the literature.

Clinical utility gene card for: poikiloderma with neutropenia.

1.5 Mutational spectrum Poikiloderma with Neutropenia (PN), a very rare autosomal recessive inherited genodermatosis first described in the Navajo tribe of Native Americans,1 has recently been associated with biallelic mutations in the C16orf57 gene.2 So far, 19 different C16orf57 mutations have been detected in 37 PN patients subjected to molecular-genetic testing. Of these 37 patients, 31 (84%) carry homozygous mutations, whereas the other six are compound heterozygous.3 All identified mutations lead to the generation of truncated and most likely non-functional C16orf57 protein. C16orf57 is a 30–50 exonuclease essential for the biogenesis of the splicing apparatus.4,5 Several classes of mutations have been identified, listed here in order of decreasing prevalence: nonsense mutations (c.232C4T, c.243G4A, c.258T4A, c.267T4A, c.415C4T, c.541C4T, c.673C4T); small out-of-frame deletions (c.176_177delG, c.179delC, c.489_492del4, c.496delA, c.531delA, c.683_893þ 1del12); and splicing alterations, including substitutions at canonical splice junctions or at splice-site consensus sequences (c.265þ 2T4G, c.266 1G4A, c.450 2A4G, c.502A4G, c.504 2A4C, c.693þ 1G4T).2,3,6–10 No missense mutations have yet been found; c.502A4G can be categorised as a splicing alteration because it leads to the excision of the fourth exon from the mature C16orf57-001 transcript.2 The most frequent recurrent mutations, c.531delA, c.496delA and c.179delC, reflect three geographical clusters. c.531delA has been recorded in seven patients from the Caucasus region, two of whom are members of unrelated Turkish families; the second most frequent mutation, c.496delA, has been detected in five patients from the Athabaskan ethnic group; and the third most frequent, c.179delC, was identified in four patients of North African origin. Considering the very low frequency of PN syndrome and the prevalence of patients with homozygous mutations, common ancestry is the hypothesis most likely to explain the recurrence of these mutations in specific ethnic groups.3

Evidence of cell fragility caused by gene kil following λ induction

Abstract Escherichia coli cells carrying λ cI857 prophage lyse 40 min after λ thermoinduction; the lysis depends on the λ genes Q , R , and S . If chloramphenicol (CAP) is added within 20 min after λ cI857 induction, an early, unproductive lysis occurs. This lysis is independent of the genes int, rex, O , P , Q , and all late genes. Instead, early lysis depends upon the kil gene. The early lysis is under the positive control of λ gene N and the negative control of gene cro . One or more events specifically connected with λ induction appear to be necessary for the occurrence of early lysis, since early lysis cannot be observed after λ infection. Induced λ kil + lysogens are more sensitive to osmotic shock than induced λ kil - lysogens. CAP-induced early lysis can be prevented in a hypertonic medium. These results suggest that induction of λ causes an osmotic fragility due to a damage of the cell envelope which requires repair; in the absence of protein synthesis the cell envelope is not repaired and cell lysis ensues.

Letter in response to “RNA processing defects of the helicase gene RECQL4 in a compound heterozygous Rothmund–Thomson patient” by Beghini et al.

‘‘We have identified the same homozygous 11-bp intronic deletion by RT-PCR analysis of RECQL4 cDNA from the RTS fraternal twins of a previously described Malaysian pedigree [Tong, 1995; Miozzo et al., 1998; Balraj et al., 2002]. A 10-bp direct repeat motif flanks the deleted bases likely accounting for the occurrence of the same mutation in different populations.’’ The 11-bp deletions reported in the two articles, Wang et al. [2002] and Balraj et al. [2002] partially overlap but are two mutations occurring in two different populations (Mexicans and Malays). As the authors suggest, the mutational mechanism is likely to be the same, due to the presence of the 10-bp direct repeat motif.

Microsatellite instability in IVS3 of murine c-fes gene: Tumor-associated rearrangement and mammalian divergence

The murine lymphomacrophage hybrids ESb, EbF1, EbF2-c4, which express c -fes constitutively, were found by Southern analysis to bear a c -fes deletion of almost 100 bp. The deleted allele was transmitted to the metastatic hybrids by their nonexpressing, poorly metastatic T-lymphoma progenitor Eb, which also has a structurally normal c -fes allele. PCR amplification and sequencing of fes cDNA spanning exons 3–5, where the deletion mapped, ruled out any involvement of coding sequences in the rearrangement. PCR amplification of the as yet unsequenced murine c -fes IVS3 and IVS4 showed they are about 50% longer than their human and feline homologs. Sequencing of IVS4 showed no difference between tumor and control DNA. Sequencing of part of the ~2600-bp IVS3 was guided by the restriction analysis of PCR products from control and hybrid DNAs. This showed that differences from the control appeared to be mainly located in the 900-bp HindIII-EcoRI fragment, localized in the middle of IVS3. As all three hybrids had the same restriction map, this fragment was sequenced in one of them (ESb). A run of >200 CA repeats was found in control DNA, and a reduction in the CAn microsatellite accounted for most of the c-fes deletion in the ESb hybrid. Interestingly, the 50% reduction in the size of human and feline c -fes IVS3 as compared with the murine homolog is mostly due to contraction of the same microsatellite.