Amedea Mencarelli

The acute promyelocytic leukemia-specific PML-RARα fusion protein inhibits differentiation and promotes survival of myeloid precursor cells

Acute promyelocytic leukemia is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. The differentiation block can be reversed by retinoic acid, which induces blast maturation both in vitro and in vivo. Acute promyelocytic leukemia is characterized by a 15;17 chromosome translocation with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and the PML gene, which encodes a putative transcription factor, on 15. A PML-RAR alpha fusion protein is formed as a consequence of the translocation. We expressed the PML-RAR alpha protein in U937 myeloid precursor cells and showed that they lost the capacity to differentiate under the action of different stimuli (vitamin D3 and transforming growth factor beta 1), acquired enhanced sensitivity to retinoic acid, and exhibited a higher growth rate consequent to diminished apoptotic cell death. These results provide evidence of biological activity of PML-RAR alpha and recapitulate critical features of the promyelocytic leukemia phenotype.

Cooperation between the RING+B1-B2 and coiled-coil domains of PML is necessary for its effects on cell survival

PML/RARα is the abnormal protein product of the Acute Promyelocytic Leukemia-specific 15;17 translocation. Both the PML and RARα components are required for the PML/RARα biological activities, namely its capacity to block differentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within specific nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARα protein contains the RING finger, two newly defined cystein/histidine-rich motifs called B-boxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring+B1-B2 and coiled-coil regions are both indispensable and sufficient to target PML to the NBs; (ii) individual deletions of the various PML domains have no effect on its growth suppressor activity; (iii) the Ring+B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sufficient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring+B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the effects of PML/RARα on survival derive from the activation of the incorporated PML sequence.

Cleft palate and ADULT phenotype in a patient with a novel TP63 mutation suggests lumping of EEC/LM/ADULT syndromes into a unique entity: ELA syndrome

Acro‐dermato‐ungual‐lacrimal‐tooth (ADULT) syndrome is a rare condition belonging to the group of ectodermal dysplasias caused by TP63 mutations. Its clinical phenotype is similar to ectrodactyly‐ectodermal dysplasia‐cleft lip/palate (EEC) and limb‐mammary syndrome (LMS), and differs from these disorders mainly by the absence of cleft lip and/or palate. We report on a 39‐year‐old patient who was found to be heterozygous for a c.401G > T (p.Gly134Val) de novo mutation of TP63. This patient had the ADULT phenotype associated with cleft palate. Our findings, rather than extend the clinical spectrum of ADULT syndrome, suggest that cleft palate can no longer be considered an element for differential diagnosis for ADULT, EEC, and LMS. Our data, added to other reports on overlapping phenotypes, support the combining of these three phenotypes into a unique entity that we propose to call “ELA syndrome,” which is an acronym of ectrodactyly‐ectodermal dysplasia‐cleft lip and palate, limb‐mammary, and ADULT syndromes.

A novel MED12 mutation: Evidence for a fourth phenotype.

Mutations of the MED12 gene have been reported mainly in males with FG (Opitz–Kaveggia), Lujan–Fryns, or X‐linked Ohdo syndromes. Recently, a different phenotype characterized by minor anomalies, severe intellectual disability (ID), and absent language was reported in female and male patients belonging to the same family and carrying a frameshift MED12 mutation (c.5898dupC). Here, we report on two brothers and their niece affected by severe and mild ID, respectively, where whole exome sequencing combined with variant analysis within a panel of ID‐related genes, disclosed a novel c.2312T>C (p.Ile771Thr) MED12 mutation. This variant, which has not been reported as a polymorphism, was not present in a third unaffected brother, and was predicted to be deleterious by five bioinformatic databases. This finding together with the phenotypic analogies shared with the carriers of c.5898dupC mutation suggests the existence of a fourth MED12‐related disorder, characterized by severe ID, absent or deficient language and, milder, clinical manifestation in heterozygotes. We have reviewed the literature on MED12 heterozygotes, their clinical manifestations, and discuss the possible biological causes of this condition.

Trisomy 2 mosaicism with caudal dysgenesis, Hirschsprung disease, and micro-anophthalmia.

Trisomy 2 Mosaicism With Caudal Dysgenesis, Hirschsprung Disease, and Micro-Anophthalmia Paolo Prontera, Gabriela Stangoni, Carmela Ardisia, Daniela Rogaia, Amedea Mencarelli, and Emilio Donti* Medical Genetics Unit, Department of Clinical and Experimental Medicine, University of Perugia and ‘‘S. Maria della Misericordia’’ Hospital, Perugia, Italy Neonatology Unit, ‘‘S. Maria della Misericordia’’ Hospital, Perugia, Italy

Encephalocraniocutaneous lipomatosis (ECCL) in a patient with history of familial multiple lipomatosis (FML).

The clinical and pathogenetic classification of encephalocraniocutaneous lipomatosis (ECCL), oculoectodermal syndrome (OES), and oculocerebrocutaneous syndrome (OCCS) and their possible relationship have been recently discussed in two issues of this journal [Hunter, 2006; Ardinger et al., 2007; Moog et al., 2007a,b]. These syndromes share common features but the presence of nevus psiloliparus (NPL) as well as major brain, skin, and eye criteria distinguishes ECCL from OES and OCCS [Hunter, 2006; Ardinger et al., 2007]. These disorders are mainly sporadic and are thought to be the result of somatic mutation(s) or the expression of rare tumor predisposition syndrome(s) [Toriello et al., 1993; Federici et al., 2004; Hunter, 2006; Moog et al., 2007b]. Although their etiologies remain unclear for all three conditions, clinical evidence suggests that the etiologic or pathogenetic pathway is similar between ECCL and OES (perhaps disorders of mosaicism) and that OCCS is separate [Hunter, 2006; Moog et al., 2007b]. Hunter [2006] applied Happle’s [1987] hypothesis of a ‘‘lethal gene surviving by mosaicism’’ to ECCL etiology; moreover Moog et al. [2007b] suggested that the mutational event could occur in an autosomal gene such as High-Mobility-Group A2 (HMGA2) encoding a factor involved in vasculogenesis, lipomagenesis and development of mesenchymal tumors [Schoenmakers et al., 1995; Ligon et al., 2005; Bartuma et al., 2007]. Here we report on a 5-year-old female patient with ECCL and familial history of multiple lipomatosis (FML) (OMIM %151900). The proposita was born at term from unrelated parents. At birth she was of normal weight (3,300 g, 50th centile) and length (49 cm, 50th centile) but showed lipomas on the face and alopecia areata. At three months an ophthalmologic evaluation revealed coloboma of iris and chorioretinitis. Motor development was normal, and she began to walk on her own when she was one year. In that period, the child developed complex partial crises (one every 4 days), and an EEG disclosed a reduction of the voltage in the right frontal and central regions. The crises did not respond to various antiepileptic drugs (levetiracetam, oxcarbazepine, lamotrigine) along with sodium valproate and their frequency was only partially reduced by the administration of phenobarbital (4–5 mg/kg/day) and valproate (31.5 mg/kg/day). At 20 months, an echocardiogram revealed a moderate tricuspid valve insufficiency causing a mild increase of the pulmonary pressure; no other congenital heart abnormalities or anomalies of the aorta were observed. At 3 years an MRI and a CT scan of brain and facial bones disclosed intracranial lipomas (along the trigeminus), leptomeningeal angiomatosis and intracranial calcification, hypoplastic corpus callosum, aracnoid cyst of the right temporal lobe, enlargement of right ventricle, microgyria of the temporal and occipital right lobes and an osteolytic lesion in the right jaw, including irregular zones of calcification, ossification and dental elements (odontogenic jaw-tumor). When she was 3 years a neuropsychiatric assessment disclosed a moderate speech delay and, although an IQ evaluation was not possible because of poor cooperation, the child did not seem to show mental retardation. At the time of our evaluation the child was 4 years and 10 months old and showed macrocephaly (OFC 54 cm, >97th centile), bilateral epibulbar dermoid, bilateral palpebral fibrolipomas, bilateral temporal subcutaneous dermolipomas, small periorbital skin tags and alopecia areata (Fig. 1). The dental arches were pointed and the areas between teeth were characterized by bone expansion. The patient met diagnostic criteria for ECCL [Hunter, 2006]. Chromosome analysis, carried out on peripheral blood lymphocytes at 500 band resolution using standard procedure, revealed a normal 46,XX karyotype. Since the clinical picture overlapped the phenotype of Bannayan–Riley–Ruvalcaba syndrome caused by

FMR1, FMR2, and SLITRK2 deletion inside a paracentric inversion involving bands Xq27.3–q28 in a male and his mother†

FMR1, FMR2, and SLITRK2 Deletion Inside a Paracentric Inversion Involving Bands Xq27.3–q28 in a Male and His Mother Simona Cavani,* Paolo Prontera, Marina Grasso, Carmela Ardisia, Michela Malacarne, Cristina Gradassi, Massimiliano Cecconi, Amedea Mencarelli, Emilio Donti, and Mauro Pierluigi Laboratory of Genetics, Galliera Hospital, Genoa, Italy Medical Genetics Unit, University and Hospital of Perugia, Perugia, Italy

Expanding the Clinical Spectrum of Sotos Syndrome in a Patient with the New “c.[5867T>A]+[=]”; “p.[Leu1956Gln]+[=]” NSD1 Missense Mutation and Complex Skin Hamartoma

Sotos syndrome is one of the most common overgrowth diseases and it predisposes patients to cancer, generally in childhood. The prevalence of this genetic disorder is 1:10,000–1:50,000, and it is characterized by wide allelic heterogeneity, with more than 100 different known mutations in the nuclear receptor-binding SET domain containing protein 1 (NSD1) gene. Most of these alterations are deletions and common micro-deletions with haploinsufficiency. Singular variants are missense mutations. The present study reports a case of a 4-year-old boy with specific clinical features of Sotos syndrome and a particular complex skin hamartoma on the right femoral side, in addition to other minor findings, such as a “café-au-lait” spot on the right hemithorax and syndactyly of the second and third right toes. NSD1 gene analysis identified a de novo missense mutation, “c.[5867T>A]+[=]”; “p.[Leu1956Gln]+[=]”, that was not previously described in the literature. This mutation was localized to the functional domain of the gene and was likely the cause of Sotos syndrome in our patient. We also compared aspects of our patient’s condition with the clinical features of tuberous sclerosis (TSC), which is an autosomal neurocutaneous syndrome caused by mutations in the TSC1/TSC2 genes. These genes control cell growth and cell survival. This disorder is characterized by hamartomas in multiple organ systems, several coetaneous abnormalities, epilepsy, and increased risk of several types of tumors.

REPORT OF A NOVEL SHOX MISSENSE VARIANT IN A BOY WITH SHORT STATURE AND HIS MOTHER WITH LERI-WEILL DYSCHONDROSTEOSIS

Heterozygous mutations in the SHOX gene or in the upstream and downstream enhancer elements are associated with 2–22% of cases of idiopathic short stature (OMIM #300582) and with 60% of cases of Leri–Weill dyschondrosteosis (OMIM #127300) with which female subjects are generally more severely affected. Approximately 80–90% of SHOX pathogenic variants are deletions or duplications, and the remaining 10–20% are point mutations that primarily give rise to missense variants. The clinical interpretation of novel variants, particularly missense variants, can be challenging and can remain of uncertain significance. Here, we describe a novel missense variant (c.1044 G>T, p.Arg118Met) in a Moroccan boy with a disproportionately short stature and without any radiological traits or bone deformities and in his mother, who had a disproportionately short stature and a Madelung deformity. This variant has not been reported to date in the updated SHOX allelic variant or Human Gene Mutation Databases nor is it listed as a polymorphism in the ExAC browser, dbSNP, or 1000G. This mutation was predicted to be deleterious by three different bioinformatics tools since it modifies an amino acid in a highly conserved DNA-binding domain of the SHOX protein. Based on this evidence, the patient was treated with recombinant human growth hormone.

Autosomal Dominant PTH Gene Signal Sequence Mutation in a Family With Familial Isolated Hypoparathyroidism

Context: Familial isolated hypoparathyroidism (FIH) is a genetically heterogeneous disorder due to mutations of the calcium‐sensing receptor (CASR), glial cells missing‐2 (GCM2), guanine nucleotide binding protein &agr;11 (GNA11), or parathyroid hormone (PTH) genes. Thus far, only four cases with homozygous and two cases with heterozygous mutations in the PTH gene have been reported. Objective: To clinically describe an FIH family and identify and characterize the causal gene mutation. Design: Genomic DNA of the family members was subjected to CASR, GCM2, GNA11, and PTH gene mutational analysis. Functional assays were performed on the variant identified. Participants: Six subjects of a three‐generation FIH family with three affected individuals having severe hypocalcemia and inappropriately low serum PTH. Results: No mutations were detected in the CASR, GCM2, and GNA11 genes. A heterozygous variant that segregated with the disease was identified in PTH gene exon 2 (c.41T>A; p.M14K). This missense variant, in the hydrophobic core of the signal sequence, was predicted in silico to impair cleavage of preproPTH to proPTH. Functional assays in HEK293 cells demonstrated much greater retention intracellularly but impaired secretion into the medium of the M14K mutant relative to wild type. The addition of the pharmacological chaperone, 4‐phenylbutyric acid, led to a reduction of cellular retention and increased accumulation in the cell medium of the M14K mutant. Conclusions: We report a heterozygous PTH mutation in an FIH family and demonstrate accumulation of the mutant intracellularly and its impaired secretion. An accurate genetic diagnosis in such hypoparathyroid patients is critical for appropriate treatment and genetic counseling.