Andrzej Poplawski

Phenotypically concordant and discordant monozygotic twins display different DNA copy-number-variation profiles.

The exploration of copy-number variation (CNV), notably of somatic cells, is an understudied aspect of genome biology. Any differences in the genetic makeup between twins derived from the same zygote represent an irrefutable example of somatic mosaicism. We studied 19 pairs of monozygotic twins with either concordant or discordant phenotype by using two platforms for genome-wide CNV analyses and showed that CNVs exist within pairs in both groups. These findings have an impact on our views of genotypic and phenotypic diversity in monozygotic twins and suggest that CNV analysis in phenotypically discordant monozygotic twins may provide a powerful tool for identifying disease-predisposition loci. Our results also imply that caution should be exercised when interpreting disease causality of de novo CNVs found in patients based on analysis of a single tissue in routine disease-related DNA diagnostics.

Somatic mosaicism for copy number variation in differentiated human tissues

Two major types of genetic variation are known: single nucleotide polymorphisms (SNPs), and a more recently discovered structural variation, involving changes in copy number (CNVs) of kilobase‐ to megabase‐sized chromosomal segments. It is unknown whether CNVs arise in somatic cells, but it is, however, generally assumed that normal cells are genetically identical. We tested 34 tissue samples from three subjects and, having analyzed for each tissue ≤10–6 of all cells expected in an adult human, we observed at least six CNVs, affecting a single organ or one or more tissues of the same subject. The CNVs ranged from 82 to 176 kb, often encompassing known genes, potentially affecting gene function. Our results indicate that humans are commonly affected by somatic mosaicism for stochastic CNVs, which occur in a substantial fraction of cells. The majority of described CNVs were previously shown to be polymorphic between unrelated subjects, suggesting that some CNVs previously reported as germline might represent somatic events, since in most studies of this kind, only one tissue is typically examined and analysis of parents for the studied subjects is not routinely performed. A considerable number of human phenotypes are a consequence of a somatic process. Thus, our conclusions will be important for the delineation of genetic factors behind these phenotypes. Consequently, biobanks should consider sampling multiple tissues to better address mosaicism in the studies of somatic disorders. Hum Mutat 0,1–7, 2008.

Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas

Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1.

Frequent genetic differences between matched primary and metastatic breast cancer provide an approach to identification of biomarkers for disease progression.

Breast cancer is a major cause of morbidity and mortality in women and its metastatic spread is the principal reason behind the fatal outcome. Metastasis-related research of breast cancer is however underdeveloped when compared with the abundant literature on primary tumors. We applied an unexplored approach comparing at high resolution the genomic profiles of primary tumors and synchronous axillary lymph node metastases from 13 patients with breast cancer. Overall, primary tumors displayed 20% higher number of aberrations than metastases. In all but two patients, we detected in total 157 statistically significant differences between primary lesions and matched metastases. We further observed differences that can be linked to metastatic disease and there was also an overlapping pattern of changes between different patients. Many of the differences described here have been previously linked to poor patient survival, suggesting that this is a viable approach toward finding biomarkers for disease progression and definition of new targets useful for development of anticancer drugs. Frequent genetic differences between primary tumors and metastases in breast cancer also question, at least to some extent, the role of primary tumors as a surrogate subject of study for the systemic disease.

The ftsZ gene of Haloferax mediterranei: sequence, conserved gene order, and visualization of the FtsZ ring

We sequenced the ftsZ gene region of the halophilic archaeon Haloferax mediterranei and mapped the transcription start sites for the ftsZ gene. The gene encoded a 363-amino-acid long FtsZ protein with a predicted molecular mass of 38 kDa and an isoelectric point of 4.2. A high level of similarity to the FtsZ protein of Haloferax volcanii was apparent, with 97 and 90% identity at the amino acid and nucleotide levels, respectively. Structural conservation at the protein level was shown by visualization of the FtsZ ring structure in H. mediterranei cells using an antiserum raised against FtsZ of H. volcanii. FtsZ rings were observed in cells in different stages of division, including cells with pleomorphic shapes and cells that appeared to be undergoing asymmetric division. Cells were also observed that displayed constriction-like invaginations in the absence of an FtsZ ring, indicating that morphological data are not sufficient to determine whether pleomorphic Haloferax cells are undergoing cell division. Both the upstream and downstream gene order in the ftsZ region was found to be conserved within the genus Haloferax. Furthermore, the downstream gene order, which includes the secE and nusG genes, is conserved in almost all euryarchaea sequenced to date. The secE and nusG genes are likely to be transcriptionally and translationally coupled in Haloferax, and this co-expression may have been a selective force that has contributed to keeping the gene cluster intact.

Altered patterns of cellular growth, morphology, replication and division in conditional-lethal mutants of the thermophilic archaeon Sulfolobus acidocaldarius.

As a basis for studing the essential cellular processes of hyperthermophilic archaea, thermosensitive mutants of Sulfolobus acidocaldarius were isolated and characterized. Exponential-phase liquid cultures were shifted to the nonpermissive temperature and growth, viability, and distributions of cell mass and DNA content were measured as a function of time after the shift. The observed phenotypes demonstrate that chromosome replication, nucleoid organization, nucleoid partition and cell division, which normally are tightly co-ordinated during cellular growth, can be inhibited or uncoupled by mutation in this hyperthermophilic archaeon.

Multiple, diffuse schwannomas in a RASopathy phenotype patient with germline KRAS mutation: a causal relationship?

To the Editor : RAS proto-oncogenes (KRAS, HRAS and NRAS ) encode GTPases, and their role in cancer, as somatic mutations, is well known (1, 2). Germline mutations in genes within the RAS/MAPK signaling pathway are responsible for a group of phenotypically overlapping developmental disorders (RASopathies) (3), including Noonan syndrome (NS) and Noonan-related disorders: NS with multiple lentigines, cardio-facio-cutaneous syndrome and Costello syndrome (CS) (4). Tumor risk in RASopathies is not yet fully established, and more studies are needed (5, 6). NS patients harboring PTPN11 germline mutations have a 3.5-fold increased risk compared to the general population (6). CS patients carrying HRAS mutations have a higher incidence (approximately 15%) of certain solid tumors, especially rhabdomyosarcomas, and a screening surveillance protocol has been proposed (7, 8). Here, we describe the presence of multiple diffuse schwannomas in a patient harboring a germline KRAS mutation (p.K5E), previously reported on (9). The proband is a 23-year-old female with clinical features compatible with a RASopathy phenotype (Fig. 1a). She was initially evaluated in our service at 8 years old. At the age of 22, she started to experience difficulty in relaxation of the hand muscles, fatigue, abdominal pain and nausea. Six months later, a superficial nodule in her forth right intercostal space was palpable. An exploratory magnetic resonance imaging (MRI) scan of her cranium, thorax and abdomen showed diffuse enlargement of all nerve roots of the spine, brachial plexus, radial, ulnar and median nerves, as well as masses on the retroperitoneum (Fig. 1b), suggesting an overgrowth of peripheral nerve sheaths, compatible with a schwannoma or neurofibroma. No brain abnormalities were found by MRI scanning. Histopathological and immunohistochemical analysis of an excised thoracic nodule were compatible with the diagnosis of schwannoma (Fig. 1c). Combining histological and MRI findings, our final diagnosis was multiple diffuse schwannomas. In otherwise healthy patients, benign tumors of the peripheral nerve sheath typically occur singly. In rare cases malignant transformation occurs. The presence of multiple schwannomas in an individual suggests an underlying tumor predisposition syndrome, more particularly neurofibromatosis type II (NF2) or schwannomatosis (SMARCB1 ) (10). Comprehensive NF2 and SMARCB1 mutation analysis was performed on DNA extracted from blood and tumor. Analysis included sequencing of the entire coding region, dosage analysis by Multiplex Ligation Probe Assay and Loss of Heterozygosity analysis using 15 microsatellite markers along chromosome 22q. No pathogenic NF2/SMARCB1 mutations were identified. A novel NF2 benign intronic variant was identified in blood and tumor: c.88615C>T. This alteration replaces a non-evolutionary conserved pyrimidine by another pyrimidine at the splice acceptor of exon 10 and was proven not to affect splicing through RNA analysis in blood, in agreement with results from in silico splice predictions. Thus, the co-occurrence of a KRAS -associated phenotype and NF2/schwannomatosis caused by mutations in NF2 or SMARCB1 genes is highly unlikely. Therefore, KRAS could be in itself the tumor predisposing gene leading to diffuse schwannomas in our patient. As it is known that SMARCB1 mutations are only found in a minority of the NF2 -negative schwannomatosis patients (11, 12), we further performed sequence analysis of all KRAS exons in eight NF2/SMARCB1 negative schwannomatosis patients, but no mutations were found, indicating that KRAS might not be a major contributing gene in NF2/SMARCB1 -negative schwannomatosis patients. However, the presence of schwannoma has been reported previously in an NS and a CS patient, though this was prior to the identification of the genes causing these disorders (13, 14). Exacerbated growth of peripheral nerve sheaths (neurofibromas) is also observed in neurofibromatosis type 1 patients carrying NF1 mutations, another tumor suppressor gene of the RAS/MAPK pathway. Activation of this pathway regulates mTOR which is phosphoinositol 3′ kinase (PI3K) dependent, a critical effector pathway involved in disease pathogenesis (15). Although functional studies are not available for the mutation p.K5E found in our patient, a different gene

Unilateral Vestibular Schwannoma and Meningiomas in a Patient with PIK3CA-Related Segmental Overgrowth: Co-occurrence of Mosaicism for Two Rare Disorders

A 28‐year‐old female with PIK3CA‐related segmental overgrowth presented with headaches. She also had a unilateral vestibular schwannoma (VS), as well as 3 small (<2 cm) meningiomas, which according to the Manchester consensus diagnostic criteria for neurofibromatosis 2 (NF2) is sufficient for a clinical diagnosis. Analysis of blood revealed a mosaic PIK3CA c.2740G>A (p.Gly914Arg) mutation, confirming the diagnosis of PIK3CA‐related overgrowth, but no mutations in NF2 were detected. Although VS has not previously been reported in PIK3CA‐related segmental overgrowth, meningiomas have, raising the question of whether this patients VS and meningiomas represent coincidental NF2 or phenotypic extension of her overgrowth syndrome. Genetic analysis of the VS revealed a heterozygous NF2 mutation c.784C>T (p.Arg262Ter) and loss of a portion of 22q, including NF2, SMARCB1, and LZTR1 genes. These results suggest that the patient has 2 different mosaic disorders, NF2 and PIK3CA‐related overgrowth. The PIK3CA mutation was also present in the VS. Confirmation of the clinical diagnosis of mosaic NF2 in this patient has implications for monitoring and highlights the possibility of co‐occurrence of mosaicism for multiple rare disorders in a single patient.

Alternative outcomes of pathogenic complex somatic structural variations in the genomes of NF1 and NF2 patients

Multiplex ligation-dependent probe amplification (MLPA) has been widely used to identify copy-number variations (CNVs), but MLPA’s sensitivity and specificity in mosaic CNV detection are largely unknown. Here, we present two mosaic deletions identified by MLPA as NF1 deletion of exons 17–21 and NF2 deletion of exons 9–10. Through cDNA analysis, genomic breakpoint-spanning PCR and Sanger sequencing, we found however both NF1 and NF2 deletions are each composed of two consecutive deletions, which cannot be differentiated by MLPA. Importantly, these consecutive deletions are most likely originating from a single genomic rearrangement and have been preserved independently in different populations of cells.