Hereditary leiomyomatosis and renal cell cancer (HLRCC)

Hereditary leiomyomatosis and renal cell cancer (HLRCC) (MIM 605839) is a recently identified autosomal dominant tumor susceptibility syndrome characterized by predisposition to benign leiomyomas of the skin and the uterus (fibroids, myomas). Susceptibility to early-onset renal cell carcinoma and uterine leiomyosarcoma is present in a subset of families. Renal cell carcinomas are typically solitary and aggressive tumors displaying papillary type 2 or collecting duct histology. The disease predisposing gene was identified as fumarate hydratase (fumarase, FH) (MIM 136850). FH encodes an enzyme that operates in the mitochondrial Krebs cycle being thus involved in cellular energy metabolism. The recent discovery of HLRCC and the predisposing gene FH has increased the present knowledge of hereditary renal cancer and enabled identification of the predisposed individuals. This review provides the present knowledge of the clinical, histopathological, and molecular features of HLRCC. Future prospects related to studies on the phenotype and molecular biology of HLRCC will also be discussed.     

Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a progressive upper-motor neurodegenerative disease. The eighth HSP locus, SPG8, is on chromosome 8p24.13. The three families previously linked to the SPG8 locus present with relatively severe, pure spastic paraplegia. We have identified three mutations in the KIAA0196 gene in six families that map to the SPG8 locus. One mutation, V626F, segregated in three large North American families with European ancestry and in one British family. An L619F mutation was found in a Brazilian family. The third mutation, N471D, was identified in a smaller family of European origin and lies in a spectrin domain. None of these mutations were identified in 500 control individuals. Both the L619 and V626 residues are strictly conserved across species and likely have a notable effect on the structure of the protein product strumpellin. Rescue studies with human mRNA injected in zebrafish treated with morpholino oligonucleotides to knock down the endogenous protein showed that mutations at these two residues impaired the normal function of the KIAA0196 gene. However, the function of the 1,159-aa strumpellin protein is relatively unknown. The identification and characterization of the KIAA0196 gene will enable further insight into the pathogenesis of HSP.     

Increasing reactive oxygen species as a therapeutic approach to treat hereditary leiomyomatosis and renal cell carcinoma

Hereditary leiomyomatosis renal cell carcinoma (HLRCC)-associated renal tumors are aggressive and tend to metastasize early. There are currently no effective forms of therapy for patients with advanced HLRCC-associated kidney cancer. We have previously shown that HLRCC cells express a high level of reactive oxygen species (ROS). In the present study we investigated the cytotoxic-effects of increasing ROS level using bortezomib in combination with cisplatin on HLRCC cells in vitro and in an in vivo xenograft model. The cytotoxic effect of several ROS inducers on FH-deficient cells was assessed by synthetic lethality. ROS inducers had a pronounced impact on the viability of FH-deficient cells. Because of its high potency, the proteasome inhibitor bortezomib was further investigated. Bortezomib induced apoptosis in vitro in HLRCC cells and inhibited HLRCC tumor growth in vivo. Bortezomib-associated cytotoxicity was highly correlated with cellular ROS level: combining bortezomib with other ROS inducers enhanced cytotoxicity, while combining bortezomib with a ROS scavenger inhibited its cytotoxic effect. Finally, HLRCC murine xenografts were treated with bortezomib and cisplatin, another ROS inducer. This regimen induced HLRCC tumor regression in vivo. These findings suggest that increasing ROS level in HLRCC above a certain threshold can induce HLRCC-tumor cell death. Increasing tumor ROS with bortezomib in combination with cisplatin represents a novel targeted therapeutic approach to treat advanced HLRCC-associated renal tumors.Key words: HLRCC, bortezomib, cisplatin, ROS, kidney cancer, FH     

Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are hamartomatous vascular malformations characterized by abnormally enlarged capillary cavities without intervening brain parenchyma. They cause seizures and cerebral hemorrhages, which can result in focal neurological deficits. Three CCM loci have been mapped, and loss-of-function mutations were identified in the KRIT1 (CCM1) and MGC4607 (CCM2) genes. We report herein the identification of PDCD10 (programmed cell death 10) as the CCM3 gene. The CCM3 locus has been previously mapped to 3q26-27 within a 22-cM interval that is bracketed by D3S1763 and D3S1262. We hypothesized that genomic deletions might occur at the CCM3 locus, as reported previously to occur at the CCM2 locus. Through high-density microsatellite genotyping of 20 families, we identified, in one family, null alleles that resulted from a deletion within a 4-Mb interval flanked by markers D3S3668 and D3S1614. This de novo deletion encompassed D3S1763, which strongly suggests that the CCM3 gene lies within a 970-kb region bracketed by D3S1763 and D3S1614. Six additional distinct deleterious mutations within PDCD10, one of the five known genes mapped within this interval, were identified in seven families. Three of these mutations were nonsense mutations, and two led to an aberrant splicing of exon 9, with a frameshift and a longer open reading frame within exon 10. The last of the six mutations led to an aberrant splicing of exon 5, without frameshift. Three of these mutations occurred de novo. All of them cosegregated with the disease in the families and were not observed in 200 control chromosomes. PDCD10, also called "TFAR15," had been initially identified through a screening for genes differentially expressed during the induction of apoptosis in the TF-1 premyeloid cell line. It is highly conserved in both vertebrates and invertebrates. Its implication in cerebral cavernous malformations strongly suggests that it is a new player in vascular morphogenesis and/or remodeling.     

Mutations in the glycosylphosphatidylinositol gene PIGL cause CHIME syndrome

CHIME syndrome is characterized by colobomas, heart defects, ichthyosiform dermatosis, mental retardation (intellectual disability), and ear anomalies, including conductive hearing loss. Whole-exome sequencing on five previously reported cases identified PIGL, the de-N-acetylase required for glycosylphosphatidylinositol (GPI) anchor formation, as a strong candidate. Furthermore, cell lines derived from these cases had significantly reduced levels of the two GPI anchor markers, CD59 and a GPI-binding toxin, aerolysin (FLAER), confirming the pathogenicity of the mutations.     

Increasing reactive oxygen species as a therapeutic approach to treat hereditary leiomyomatosis and renal cell carcinoma

Hereditary leiomyomatosis renal cell carcinoma (HLRCC)-associated renal tumors are aggressive and tend to metastasize early. There are currently no effective forms of therapy for patients with advanced HLRCC-associated kidney cancer. We have previously shown that HLRCC cells express a high level of reactive oxygen species (ROS). In the present study we investigated the cytotoxiceffects of increasing ROS level using bortezomib in combination with cisplatin on HLRCC cells in vitro and in an in vivo xenograft model. The cytotoxic effect of several ROS inducers on FH-deficient cells was assessed by synthetic lethality. ROS inducers had a pronounced impact on the viability of FH-deficient cells. Because of its high potency, the proteasome inhibitor bortezomib was further investigated. Bortezomib induced apoptosis in vitro in HLRCC cells and inhibited HLRCC tumour growth in vivo. Bortezomib-associated cytotoxicity was highly correlated with cellular ROS level: combining bortezomib with other ROS inducers enhanced cytotoxicity, while combining bortezomib with a ROS scavenger inhibited its cytotoxic effect. Finally, HLRCC murine xenografts were treated with bortezomib and cisplatin, another ROS inducer. This regimen induced HLRCC tumour regression in vivo. These findings suggest that increasing ROS level in HLRCC above a certain threshold can induce HLRCC-tumor cell death. Increasing tumor ROS with bortezomib in combination with cisplatin represents a novel targeted therapeutic approach to treat advanced HLRCC-associated renal tumors.     

Mutations in the novel mitochondrial protein REEP1 cause hereditary spastic paraplegia type 31

Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous diseases that affect the upper motor neurons and their axonal projections. For the novel SPG31 locus on chromosome 2p12, we identified six different mutations in the receptor expression-enhancing protein 1 gene (REEP1). REEP1 mutations occurred in 6.5% of the patients with HSP in our sample, making it the third-most common HSP gene. We show that REEP1 is widely expressed and localizes to mitochondria, which underlines the importance of mitochondrial function in neurodegenerative disease.     

Mutations in the APC tumour suppressor gene cause chromosomal instability

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.     

Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome

Craniofrontonasal syndrome (CFNS) is an X-linked craniofacial disorder with an unusual manifestation pattern, in which affected females show multiple skeletal malformations, whereas the genetic defect causes no or only mild abnormalities in male carriers. Recently, we have mapped a gene for CFNS in the pericentromeric region of the X chromosome that contains the EFNB1 gene, which encodes the ephrin-B1 ligand for Eph receptors. Since Efnb1 mutant mice display a spectrum of malformations and an unusual inheritance reminiscent of CFNS, we analyzed the EFNB1 gene in three families with CFNS. In one family, a deletion of exons 2-5 was identified in an obligate carrier male, his mildly affected brother, and in the affected females. In the two other families, missense mutations in EFNB1 were detected that lead to amino acid exchanges P54L and T111I. Both mutations are located in multimerization and receptor-interaction motifs found within the ephrin-B1 extracellular domain. In all cases, mutations were found consistently in obligate male carriers, clinically affected males, and affected heterozygous females. We conclude that mutations in EFNB1 cause CFNS.     

Mutations in a p-type ATPase gene cause axonal degeneration

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wld(s) and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.     

Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation

We have identified truncating mutations in the human DLG3 (neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations.     

Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy associated with progressive distal sensory loss and severe ulcerations. Mutations in the first subunit of the enzyme serine palmitoyltransferase (SPT) have been associated with HSAN-I. The SPT enzyme catalyzes the first and rate-limiting step in the de novo sphingolipid synthesis pathway. However, different studies suggest the implication of other genes in the pathology of HSAN-I. Therefore, we screened the two other known subunits of SPT, SPTLC2 and SPTLC3, in a cohort of 78 HSAN patients. No mutations were found in SPTLC3, but we identified three heterozygous missense mutations in the SPTLC2 subunit of SPT in four families presenting with a typical HSAN-I phenotype. We demonstrate that these mutations result in a partial to complete loss of SPT activity in vitro and in vivo. Moreover, they cause the accumulation of the atypical and neurotoxic sphingoid metabolite 1-deoxy-sphinganine. Our findings extend the genetic heterogeneity in HSAN-I and enlarge the group of HSAN neuropathies associated with SPT defects. We further show that HSAN-I is consistently associated with an increased formation of the neurotoxic 1-deoxysphinganine, suggesting a common pathomechanism for HSAN-I.     

Purification and properties of a thermostable fumarate hydratase from the archaeobacterium Sulfolobus solfataricus

Fumarate hydratase (EC 4.2.1.2) from the extremely thermophilic archaeobacterium Solfolobus solfataricus has been purified to homogeneity by a rapid purification procedure using affinity chromatography and high-performance size-exclusion chromatography, and the enzyme's physical and biochemical properties have been determined. The native enzyme has a molecular mass of 170 kDa and is composed of identical subunits with a molecular mass of 45 kDa, thus indicating a tetrameric structure similar to fumarases isolated from other organisms. The enzyme was active at temperatures ranging from 40 degrees C to 90 degrees C, with a maximum activity at 85 degrees C. The pH optimum for generation of fumarate was found to be pH 8.0. The enzyme showed high stability to denaturation by heat and organic solvents.     

Mutations in neuromusculin,a gene encoding a cell adhesion molecule,cause nervous system defects

The Drosophila neuromusculin (nrm) gene encodes an immunoglobulin-like (Ig-like) cell adhesion molecule expressed in the precursors of the embryonic peripheral nervous system (PNS), in the midline precursors of the central nervous system (CNS), and in muscles. During the initial phases of CNS axonogenesis, nrm is expressed in cells involved in the development of commissures and longitudinal tracts. Mutations which alter expression of nrm mRNAs cause aberrant development of commissures and longitudinal axon pathways. Defects in the PNS and muscles of nrm mutants are also observed. In most nrm embryos, abnormal development can be detected in a subset of abdominal segments; however, in approximately 1 of 10 nrm embryos, the defects extend to all segments. Herein, we present evidence that nrm plays an important role in early morphogenesis, possibly by mediating or facilitating inductive cell contacts and movements.