Activating Germline Mutations Research Articles

Ruling in a Suspect: The Role ofAP2S1Mutations in Familial Hypocalciuric Hypercalcemia Type 3

Ruling in a Suspect: The Role of<i>AP2S1</i>Mutations in Familial Hypocalciuric Hypercalcemia Type 3

Germline JAK2 Mutations In The Kinase Domain Are Responsible For Hereditary Thrombocytosis and Are Resistant To JAK2 and HSP90 Inhibitors

The main molecular basis of essential thrombocytemia and hereditary thrombocytosis are acquired and germline activating mutations affecting the thrombopoietin signaling axis, respectively. We have identified two families with hereditary thrombocytosis presenting novel heterozygous germline mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, while the other presents two JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of JAK2 R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-MPL but not of Ba/F3-EPOR cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed a TPO-independent phosphorylation of STAT1. JAK2 R867Q and S755R/R938Q, compared to JAK2 V617F, had significantly longer half-lives correlating with an increased MPL cell surface expression. Moreover, these mutations conferred a resistance to JAK2 and HSP90 inhibitors compared to JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL, while inducing resistance to clinically available JAK2 inhibitors. Disclosures:No relevant conflicts of interest to declare.

Decreased bone mineral density in Costello syndrome

IntroductionCostello syndrome (CS) is a multisystemic disorder characterized by postnatal reduced growth, facial dysmorphism, cardiac defects, cognitive impairment, skin and musculo-skeletal anomalies, and predisposition to certain cancers. CS is caused by activating germline mutations in the HRAS proto-oncogene. Similar to what is observed in other RASopathies, CS causative HRAS mutations promote enhanced signal flow through the RAF–MEK–ERK and PI3K–AKT signaling cascades. While decreased bone mineralization has been documented in other RASopathies, such as neurofibromatosis type 1 and Noonan syndrome, systematic studies investigating bone mineral density (BMD) are lacking in CS. Materials and methodsDual-energy X-ray absorptiometry (DXA) was utilized to assess BMD and body composition (fat and fat-free mass) in a cohort of subjects with molecularly confirmed diagnosis of CS (n=9) and age-matched control individuals (n=29). Using general linear regression, subtotal body (total body less head), lumbar, femoral neck and femur BMD parameters were compared considering age, sex, body mass index (BMI) and Tanner stage. Blood and urine biomarkers of bone metabolism were also assessed. ResultsAll individuals with CS showed significantly lower mean values of subtotal, lumbar and femoral neck BMD compared to the control group (p≤0.01). Similarly, mean total body mass and fat-free mass parameters were lower among the CS patients than in controls (p<0.01). Low 25-OH vitamin D concentration was documented in all individuals with CS, with values below the reference range in two patients. No significant correlation between vitamin D levels and BMD parameters was observed. DiscussionCS belongs to a family of developmental disorders, the RASopathies, that share skeletal defects as a common feature. The present data provide evidence that, similar to what is recently seen in NF1 and NS, bone homeostasis is impaired in CS. The significant decrease in BMD and low levels of vitamin D documented in the present cohort, along with the risk for pathologic fractures reported in adult individuals with CS, testifies the requirement for a preventive treatment to alleviate evolutive complications resulting from dysregulated bone metabolism.

A girl with two syndromes: Turner syndrome and Costello syndrome. A case history

Turner syndrome (TS, OMIM#300706) is a well-defined chromosomal disorder characterized by short stature, gonadal dysgenesis (lack of spontaneous pubertal development), and somatic stigmata [Nielsen and Wohlert, 1991] associated with complete or partial absence of the second X chromosome. Patients with a small distal short arm deletion (Xp-) including the SHOX gene frequently have short stature and other TS associated skeletal anomalies, but most are at low risk of ovarian failure and generally should not be diagnosed with TS if band Xp22.3 is not deleted [Ross et al., 2001]. Costello syndrome (CS; OMIM 218040) is a rare autosomal dominant complex developmental disorder involving characteristic craniofacial features, failure to thrive, developmental delay, cardiac and skeletal anomalies, and a predisposition to develop neoplasia [reviewed by Gripp and Lin, 2009]. It is caused by activating germline mutations in the HRAS gene, encoding an important protein in the RAS-MAPK signaling pathway. We report on a Caucasian girl with a deletion of a short arm of chromosome X and Costello syndrome. She was the first child born to non-consanguineous parents at 35 weeks’ gestation by cesarean because of fetal distress on heart rate monitor. No polyhydramnios was reported. Her birth weight was 2200 g (10th centile), length was 46 cm (50th centile), and head circumference was 32 cm (50th centile). Soon after birth, she was noted to have somedysmorphic features, although the only information recorded was that of low-set ears and hypotonia. Chromosome analysis performed using peripheral blood leukocytes showed a karyotype of 46,X,del(X)(p21.2). Turner syndromewas diagnosed [Ross et al., 2001; Bondy, 2007]. Parental chromosomes were normal. Echocardiogram performed in the neonatal period was normal and electrocardiography showed first degree AV block. The neonatal coursewas complicatedbyhypoglycemia, intracranial hemorrhage, and severe anemia that required two blood transfusions. She received parenteral nutrition until the age of 8 days, and due to poor breastfeeding she required additional nutrition with nasogastric tube for several days. After discharge she was bottle fed at home and systematically gained weight. At the age of 1 year she had macrocephaly, a short, webbed neck, hypertelorism, bilateral epicathal folds,widely spacednipples, deepplantar andpalmar creases, and hyperconvex nails, but no further genetic studies were performed. Her psychomotor development was delayed. She sat at 1 year, started to walk at 2 years, and spoke her first words at 1.5 years of age. At the age of 7 years growth hormone (GH) replacement therapy was started and continued until age 13 years. Her growth improved from the 5th to over 95th centile on Turner girls growth charts (Fig. 1). At 9 years of age, due to features of spontaneous puberty (Tanner breast stage II, pubic hair stage I) and results of hormonal studies, gonadotropin-releasing hormone agonist therapy (Triptorelin) was started and continued for 2 years. The echocardiogramat8 years of agewasnormal, but at 9 years of age there was concentric left ventricular (LV) hypertrophy with a peak LV pressure gradient of 58mm of mercury. Therapy with a non-selective beta blocker (propranolol)was started and continued with a selective B1 blocker (metoprolol). Although this was an unusual phenotype for Turner syndrome, she was not referred to the genetic department until she was 13 years old. She exhibited striking dysmorphic features including relative macrocephaly, coarse face with thick lips (Fig. 2A,B), hypertelorism with downslanting palpebral fissures, macrostomia, macroglossia, increased skin pigmentation, wrinkled, loose skin of hands (Fig. 3A,B) and feet, hyperconvex and uplifted nails, perioral and nasal papillomata (Fig. 2), joint laxity, sparse eyebrows, curly and sparse hair. Her

Abstract SY25-03: Host and tumor genetic architecture influence clinically relevant oncogenic pathways in neuroblastoma

Abstract Cancer research is at a critical crossroads as genomic technologies have enabled potentially paradigm-shifting discoveries in host genetic alterations that lead to cancer, as well as the major oncogenic drivers that somatically arise and shape the ultimate tumor phenotype. Approaches such as genome-wide association studies (GWAS) to discover heritable susceptibility variants have largely occurred in parallel with tumor profiling efforts designed to discover somatically acquired mutations. We see tremendous opportunity in integrating these efforts under the broad hypothesis that the host's genetic blueprint not only influences susceptibility to cancer, but also significantly influences the cancer phenotype at diagnosis and the pathways upon which the cancer is dependent. By focusing our efforts on the pediatric cancer neuroblastoma, we are directly addressing an important childhood malignancy that continues to exact disproportionate morbidity and mortality. However, we have also shown that this disease also serves as a remarkable model for the study of human cancers in general, and we think that lessons learned here are likely to be more broadly applicable as we are addressing the concept that somatic mutations alone will not define the entire spectrum of oncogenic vulnerabilities in cancer cells. Until recently, the etiology of neuroblastoma was unknown. By studying the 1% of neuroblastoma cases that occur within families, we have discovered ALK and PHOX2B as the two major hereditary predisposition genes.1,2 In parallel, by studying the 99% of cases that occur sporadically in our ongoing GWAS, for which we have now analyzed 4,000 cases and 10,000 controls, we have shown that normal genetic variation at multiple loci influences neuroblastoma susceptibility.3-7 We have several other recently validated associations that are not yet published, including replicated signals in LIN28B and HACE1. Significant lessons from the GWAS to date include: 1) Neuroblastoma is a genetic disease; 2) every signal identified to date is intragenic; 3) associations are enriched in phenotypic subsets; and 4) many of the genes identified by the neuroblastoma GWAS not only influence malignant transformation of a developing neuroblastic cell, but also contribute to the maintenance of the malignant phenotype. For example, SNP variations in the LMO1 gene are highly associated with overexpression of this transcriptional co-factor, and genetic manipulation of expression shows that it functions as an oncogenic driver in a genotype specific manner.7 In other words, our data suggest that subjects harboring LMO1 risk alleles are much more likely to have tumors dependent on LMO1-influenced transcriptional regulation suggesting that oncogene addiction is also hardwired in the heritable genome. In parallel, we and others have shown that somatic activating mutations in the ALK kinase domain occur in ∼10% of sporadic cases.2,8-10 To seek other potential somatically acquired mutations that may be therapeutically relevant in the neuroblastoma coding genome, we have resequenced 99 high-risk neuroblastoma cases using a combination of whole exome, genome and transcriptome sequencing. Tumors contained a median 0.56 non-silent mutations per megabase of coding DNA, one of the lowest rates reported in cancer to date. ALK was the most commonly mutated gene (9%), and no other gene showed mutation frequency rates &amp;gt;5%. These data clearly indicate that somatic mutation in neuroblastoma is relatively rare, leading to the concept that other mechanisms, such as host polymorphic variation or germline mutations in key regulatory regions, play an important role in tumorigenesis. Ongoing work to be discussed at the symposium include efforts to identify and validate rare variants in the host genome that impact neuroblastoma susceptibility, understanding the functional relevance of DNA variation in the developing neuroblastoma and in tumor tissues, completion of our cataloguing of somatic mutations in high-risk disease, and efforts to understand the impact of non-coding DNA alterations in the host in tumor genomes on neuroblastoma clinical phenotype. Efforts to translate these efforts into deliverables for the clinic will be emphasized.

Patients with RET D631Y Mutations Most Commonly Present with Pheochromocytoma and not Medullary Thyroid Carcinoma

Multiple endocrine neoplasia type 2a results from an activating germline mutation in the RET proto-oncogene. Carriers of a RET mutation are at risk of medullary thyroid carcinoma, pheochromocytoma, and primary hyperparathyroidism. Most individuals with multiple endocrine neoplasia type 2a eventually develop medullary thyroid carcinoma and as there is a strong genotype-phenotype correlation, guidelines have been established as to the age recommended for prophylactic thyroidectomy. However for rare mutations in the RET proto-oncogene there is insufficient evidence to provide guidance as to the risk of medullary thyroid carcinoma. We present a family with the rare RET mutation, D631Y in which the proband initially presented with a pheochromocytoma, and review the available literature pertaining to this mutation. In 83% of index cases, pheochromocytoma was the presenting feature and only 37% of adult germline mutation carriers have developed medullary thyroid carcinoma, none of whom have been reported to have nodal or metastatic disease. Patients with a D631Y RET mutation typically present with pheochromocytoma and medullary thyroid carcinoma appears to occur with a later onset than reported with other RET mutations. Based on the current literature we recommend performing prophylactic total thyroidectomy by age 12 years for D631Y carriers although this recommendation may need to be reviewed as additional data becomes available.

Molecular description of non-autoimmune hyperthyroidism at a neonate caused by a new thyrotropin receptor germline mutation

BackgroundConstitutively activating germline mutations in the thyrotropin receptor (TSHR) gene result in non-autoimmune hyperthyroidism and can be transmitted as a dominant trait or occur sporadically. These mutations are mostly located in the serpentine part of this G-protein coupled receptor.MethodsSequencing exon 9 and 10 of the thyrotropin receptor gene in a two months old patient identified a mutation which was functionally characterized after transient transfection into COS-7 cells. Cell surface localization was investigated by an ELISA approach and for signalling properties we measured cAMP by alpha screen technology for Gs/adenylyl cyclase activation and use a reporter gene assay for determination of Gq/11 phospholipase C-β activation.ResultsWe detected a heterozygous mutation in the first extracellular loop of the TSHR gene leading to an exchange of an isoleucine residue for asparagine at amino acid position 486 (I486N). Cell surface localization was reduced to 51% of wild-type TSHR. Functional characterization of the mutant receptor revealed constitutive activation of the Gs/adenylyl cyclase pathway, in contrast basal activity of the Gq/11 pathway was comparable to the wild-type. The bovine TSH-induced cAMP accumulation was slightly reduced, but IP3 signaling was impaired.ConclusionWe identified a new TSHR germline mutation (I486N) in a neonate with non-autoimmune sporadic hyperthyroidism. The mutation is located at the extracellular loop 1 and exhibits an increase in basal cAMP accumulation, but unexpectedly impairs the capability for TSH induced Gq mediated signaling. The TSHR homology model suggests isoleucine 486 as a potential key-player for induction of signal transduction by an interplay with further activation sensitive extracellular parts.

Subclinical Nonautoimmune Hyperthyroidism in a Family Segregates with a Thyrotropin Receptor Mutation with Weakly Increased Constitutive Activity

Subclinical hyperthyroidism is usually associated with Graves' disease or toxic nodular goiter. Here we report a family with hereditary subclinical hyperthyroidism caused by a constitutively activating germline mutation of the thyrotropin receptor (TSHR) gene. The proband was a 64-year-old Japanese woman who presented with a thyroid nodule and was found to be euthyroid with a suppressed serum TSH. The nodule was not hot. Although antibodies to thyroid peroxidase and thyroglobulin antibodies were present, TSHR antibodies were not detected by TSH-binding inhibition or by bioassay. Two of her middle-aged sons, but not her daughter, also had subclinical hyperthyroidism without TSHR antibodies. Without therapy, the clinical condition of the affected individuals remained unchanged over 3 years without development of overt hyperthyroidism. A novel heterozygous TSHR point mutation causing a glutamic acid to lysine substitution at codon 575 (E575K) in the second extracellular loop was detected in the three family members with subclinical hyperthyroidism, but was absent in her one daughter with normal thyroid function. In vitro functional studies of the E575K TSHR mutation demonstrated a weak, but significant, increase in constitutive activation of the cAMP pathway. Although hereditary nonautoimmune overt hyperthyroidism is very rare, TSHR activating mutations as a cause of subclinical hyperthyroidism may be more common and should be considered in the differential diagnosis, especially if familial.

Can a familial gastrointestinal tumour syndrome be allelic with Waardenburg syndrome?

Familial gastrointestinal stromal tumours (GISTs) are rare but otherwise well-characterized tumour syndromes, most commonly occurring on a background of germline-activating mutations in the tyrosine kinase receptor c-KIT. The associated clinical spectrum reflects the constitutive activation of this gene product across a number of cell lines, generating gain-of-function phenotypes in interstitial cells of Cajal (GIST and dysphagia), mast cells (mastocytosis) and melanocytes (hyperpigmentation). We report a three-generation kindred harbouring a c-KIT germline-activating mutation resulting in multifocal GISTs, dysphagia and a complex melanocyte hyperpigmentation and hypopigmentation disorder, the latter with features typical of those observed in Waardenburg type 2 syndrome (WS2F). Sequencing of genes known to be causative for WS [microphthalmia transcription factor (MITF), Pax3, Sox10, SNAI2 ] failed to show any candidate mutations to explain this complex cutaneous depigmentation phenotype. Our case report conclusively expands the clinical spectrum of familial GISTs and shows a hitherto unrecognized link to WS. Possible mechanisms responsible for this novel cause of WS2F will be discussed.

Identification of Two Novel Mutations in the RET Proto-Oncogene in the Same Family

Activating germline mutations of the RET gene cause multiple endocrine neoplasia type 2 and familial medullary thyroid carcinoma (FMTC), conditions that are inherited in an autosomal dominant manner. In addition, somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic (nonfamilial) MTC cases. We describe a Greek family with two novel likely pathogenic sequence variants of the RET gene. The first is a C to T transition at position 2458 (c.2458C>T) that causes an arginine to cysteine substitution (p.R820C) in exon 14 in the intracellular region of the kinase. This sequence variant was identified in an apparently healthy woman who had a recently deceased sister with confirmed aggressive MTC (age of onset 37 years). To assess the pathogenicity of this novel missense sequence variant, screening was performed on all available relatives: her two sons, the mother, and a second sister, including an MTC tumor sample from the deceased sister of the proband. At the time of the investigation, no clinical symptoms suggestive of multiple endocrine neoplasia type 2 or MTC were present in any of the individuals screened. The c.2458C>T transition was found in one son, the living sister, and the mother. Interestingly, it was not present in the tumor sample from the deceased sister. Instead, an in-frame deletion of 54 nt in exon 10 resulting in a protein missing 18 amino acids from I590 to G608 (c.1766_1819del 54) was found. Both genetic alterations were present in heterozygous state. These data suggest that the novel in-frame deletion was the disease-causing mutation in the deceased sister. The effect of the 2458C>T mutation on the activity of the kinase is under investigation.

Four novel RET germline variants in exons 8 and 11 display an oncogenic potential in vitro

Most germline-activating mutations of the RET proto-oncogene associated with inherited medullary thyroid cancer (MTC) are localized in exons 10, 11 and 13-15. Four novel RET variants, located in the extracellular domain (p.A510V, p.E511K and p.C531R) coded by exon 8 and in the intracellular juxtamembrane region (p.K666N) coded by exon 11, were identified on the leukocyte DNA from apparently sporadic cases. Plasmids carrying Ret9-wild-type (Ret9-WT), Ret9-C634R and all Ret9 variants were transfected, and the phosphorylation levels of RET and ERK were evaluated by western blot analyses. The transforming potentials were assessed by the focus formation assay. The p.A510V, p.E511K and p.C531R variants were found to generate RET and ERK phosphorylation levels and to have a transforming activity higher than that of Ret9-WT variant, but lower than that of Ret9-C634R variant. Differently, the p.K666N variant, located immediately downstream of the transmembrane domain, and involving a conserved residue, displayed high kinase and transforming activities. Computational analysis predicted non-conservative alterations in the mutant proteins consistent with putative modifications of the receptor conformation. The molecular analyses revealed an oncogenic potential for all the novel germline RET variants. Therefore, the prevalence of exon 8 genomic variations with an oncogenic potential may be higher than previously thought, and the analysis of this exon should be considered after the exclusion of mutations in the classical hotspots. In addition, on the basis of these functional data, it is advisable to extend the genetic screening to all the first-degree relatives of the MTC patients, and to perform a strict follow-up of familial carriers.

The gene for the thyrotropin receptor (TSHR) as a candidate gene for congenital hypothyroidism with thyroid dysgenesis.

According to the central role of the TSH receptor for thyroid function and growth the gene for the TSH receptor is a possible candidate gene for mutations which result in an impairment of thyroid growth and function (Vassart and Dumont 1992). First evidence for the role of TSH receptor defects in the pathogenesis of congenital thyroid disorders was elucidated by the presence of activating germline mutations leading to congenital hyperthyroidism (Duprez et al., 1994). After the finding of partial loss-of-function mutations leading to hyperthyrotropinemia (Sunthornthepvarakul et al., 1995) it was speculated that a more severe phenotype with hypothyroidism and hypoplasia of the gland (thyroid dysgenesis) would be the result, if complete loss-of-function mutations like the isoleucine167 to asparagine mutation would occur in a homozygote or compound heterozygote state. The screening of TSHR gene mutations by SSCP in a well defined cohort of 100 children with congenital hypothyroidism (CH), diagnosed and followed since 1978 in the Childrens Hospital of Berlin, revealed one patient with hypoplasia of the thyroid to be positive for two compound heterozygote inactivating mutations of the TSHR gene, indicating thereby that the clinical approach to define phenotypes of interest could be helpful to understand the fundamental process of thyroid development.

Medullary thyroid carcinoma and biomarkers: past, present and future

The clinical management of patients with persistent or recurrent medullary thyroid carcinoma (MTC) is still under debate, because these patients either have a long-term survival, due to an indolent course of the disease, or develop rapidly progressing disease leading to death from distant metastases. At this moment, it cannot be predicted what will happen within most individual cases. Biomarkers, indicators which can be measured objectively, can be helpful in MTC diagnosis, molecular imaging and treatment, and/or identification of MTC progression. Several MTC biomarkers are already implemented in the daily management of MTC patients. More research is being aimed at the improvement of molecular imaging techniques and the development of molecular systemic therapies. Recent discoveries, like the prognostic value of plasma calcitonin and carcino-embryonic antigen doubling-time and the presence of somatic RET mutations in MTC tissue, may be useful tools in clinical decision making in the future. In this review, we provide an overview of different MTC biomarkers and their applications in the clinical management of MTC patients.

Noonan syndrome cardiac defects are caused by PTPN11 acting in endocardium to enhance endocardial-mesenchymal transformation

Noonan syndrome (NS), the most common single-gene cause of congenital heart disease, is an autosomal dominant disorder that also features proportionate short stature, facial abnormalities, and an increased risk of myeloproliferative disease. Germline-activating mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, cause about half of NS cases; other causative alleles include KRAS, SOS1, and RAF1 mutants. We showed previously that knock-in mice bearing the NS mutant Ptpn11(D61G) on a mixed 129S4/SvJae X C57BL6/J background exhibit all major NS features, including a variety of cardiac defects, with variable penetrance. However, the cellular and molecular mechanisms underlying NS cardiac defects and whether genetic background and/or the specific NS mutation contribute to the NS phenotype remained unclear. Here, using an inducible knock-in approach, we show that all cardiac defects in NS result from mutant Shp2 expression in the endocardium, not in the myocardium or neural crest. Furthermore, the penetrance of NS defects is affected by genetic background and the specific Ptpn11 allele. Finally, ex vivo assays and pharmacological approaches show that NS mutants cause cardiac valve defects by increasing Erk MAPK activation, probably downstream of ErbB family receptor tyrosine kinases, extending the interval during which cardiac endocardial cells undergo endocardial-mesenchymal transformation. Our data provide a mechanistic underpinning for the cardiac defects in this disorder.

Expanding clinical spectrum of non‐autoimmune hyperthyroidism due to an activating germline mutation, p.M453T, in the thyrotropin receptor gene

To describe clinical and genetic features of a Thai family with non-autoimmune hyperthyroidism (NAH) caused by an activating germline mutation in the thyrotropin receptor (TSHR) gene. Three affected individuals from the same family (a father and his two children) were studied. Clinical and imaging findings were reviewed and compared. GENETIC ANALYSIS: Genomic DNA was extracted from peripheral blood leukocytes and mutation analysis of the entire coding sequence of the TSHR gene was performed in both children and their parents by direct DNA sequencing. A heterozygous germline T to C transition in exon 10 of the TSHR gene (c.1358T-->C) resulting in the substitution of methionine (ATG) by threonine (ACG) at codon 453 (p.M453T) was identified in the father and his two children. They presented with different clinical severity and variable age of onset. In addition to hyperthyroidism, ventriculomegaly and bilateral shortening of the fifth metacarpal bones and the middle phalanges of the fifth fingers were consistently found in all affected individuals. Ventriculomegaly and bilateral shortening of the fifth metacarpal bones and the middle phalanges of the fifth fingers might be characteristic features of NAH because of an activating TSHR germline mutation. In addition, the shortening of the middle phalanges of the fifth fingers has never been previously described, expanding the phenotypic spectrum of the disease.

P18 is a tumor suppressor gene involved in human medullary thyroid carcinoma and pheochromocytoma development

In multiple endocrine neoplasia syndrome Type 2 (MEN2), medullary thyroid carcinoma (MTC) and pheochromocytoma (PC) are associated with hereditary activating germ-line mutations in the RET proto-oncogene. Also in a large percentage of sporadic MTCs and PCs, somatic RET mutations appear to be involved in tumor formation. In one single MEN2 family an extensive variety in disease expression may be observed, indicating that additional genetic events are responsible for progression of the disease towards a more aggressive phenotype. However, these additional mutations in both hereditary and sporadic MTC and PC development are largely unknown. Here, we show for the first time the presence of somatic mutations in the cell cycle regulator P18 in human RET-associated MTCs and PCs. Each of these mutations causes an amino acid substitution in the cyclin dependent kinase-interacting region of P18(INK4C). Since these mutations partly inhibited P18(INK4C) function and reduced its stability, our findings implicate P18 as a tumor suppressor gene involved in human MTC and PC development.

Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma

Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.

Unregulated smooth-muscle myosin in human intestinal neoplasia

A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia.

A family with a novel TSH receptor activating germline mutation (p.Ala485Val)

Autosomal dominant nonautoimmune hyperthyroidism (ADNAH) is caused by gain of function mutations in the TSH receptor (TSHr) gene and characterized by toxic thyroid hyperplasia with a variable age of onset in the absence of thyroid antibodies and clinical symptoms of autoimmune thyroid disease in at least two generations. We report here a Turkish family with a novel TSHr gene mutation with distinct features all consistent with ADNAH. Thyroid function tests of the proband were as follows: free T3: 13.1 pg/ml (N: 1.8-4.6); free T4: 5.1 ng/dl (N: 0.9-1.7); TSH: 0.01 microIU/ml (N: 0.2-4.2); and TSH receptor antibody: 2 IU/ml (N: 0-10). A heterozygous missense mutation in exon 10 of the TSHr gene (c.1454C>T) resulting in the substitution of valine for alanine at codon 485 (p.Ala485Val) was found in the father and his son and daughter. This mutation had arisen de novo in the father. Functional studies of the novel TSHr germline mutation demonstrated a higher constitutive activation of adenyl cyclase than wild type without any effect on phospholipase C activity. In conclusion, our data indicate that gain of function germline mutations in the TSHr gene should be investigated in families with members suffering from thyrotoxicosis and progressive growth of goiter, but without clinical and biochemical evidence of autoimmune thyroid disease. In addition, patients harboring the same mutation of the TSHr gene may show wide phenotypic variability with respect to the age at onset, and severity of hyperthyroidism and thyroid growth.

NRAS mutation causes a human autoimmune lymphoproliferative syndrome

The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization, and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous, and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4(-), CD8(-) alphabeta T cells. Mutations in ALPS typically affect CD95 (Fas/APO-1)-mediated apoptosis, one of the extrinsic death pathways involving TNF receptor superfamily proteins, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair CD95-mediated apoptosis. The increase in active, GTP-bound NRAS augments RAF/MEK/ERK signaling, which markedly decreases the proapoptotic protein BIM and attenuates intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects. Our observations on the effects of NRAS activation indicate that RAS-inactivating drugs, such as farnesyltransferase inhibitors should be examined in human autoimmune and lymphocyte homeostasis disorders.