Adriana Carando

RPS19 mutations in patients with Diamond-Blackfan anemia†

Diamond‐Blackfan anemia (DBA) is an inherited disease characterized by pure erythroid aplasia. Thirty percent (30%) of patients display malformations, especially of the hands, face, heart, and urogenital tract. DBA has an autosomal dominant pattern of inheritance. De novo mutations are common and familial cases display wide clinical heterogeneity. Twenty‐five percent (25%) of patients carry a mutation in the ribosomal protein (RP) S19 gene, whereas mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 are rare. These genes encode for structural proteins of the ribosome. A link between ribosomal functions and erythroid aplasia is apparent in DBA, but its etiology is not clear. Most authors agree that a defect in protein synthesis in a rapidly proliferating tissue, such as the erythroid bone marrow, may explain the defective erythropoiesis. A total of 77 RPS19 mutations have been described. Most are whole gene deletions, translocations, or truncating mutations (nonsense or frameshift), suggesting that haploinsufficiency is the basis of DBA pathology. A total of 22 missense mutations have also been described and several works have provided in vitro functional data for the mutant proteins. This review looks at the data on all these mutations, proposes a functional classification, and describes six new mutations. It is shown that patients with RPS19 mutations display a poorer response to steroids and a worse long‐term prognosis compared to other DBA patients. Hum Mutat 29(7), 911–920, 2008.

Diamond-Blackfan anemia: genotype-phenotype correlations in Italian patients with RPL5 and RPL11 mutations

Background Diamond-Blackfan anemia is a rare, pure red blood cell aplasia of childhood due to an intrinsic defect in erythropoietic progenitors. About 40% of patients display various malformations. Anemia is corrected by steroid treatment in more than 50% of cases; non-responders need chronic transfusions or stem cell transplantation. Defects in the RPS19 gene, encoding the ribosomal protein S19, are the main known cause of Diamond-Blackfan anemia and account for more than 25% of cases. Mutations in RPS24, RPS17, and RPL35A described in a minority of patients show that Diamond-Blackfan anemia is a disorder of ribosome biogenesis. Two new genes (RPL5, RPL11), encoding for ribosomal proteins of the large subunit, have been reported to be involved in a considerable percentage of patients. Design and Methods In this genotype-phenotype analysis we screened the coding sequence and intron-exon boundaries of RPS14, RPS16, RPS24, RPL5, RPL11, and RPL35A in 92 Italian patients with Diamond-Blackfan anemia who were negative for RPS19 mutations. Results About 20% of the patients screened had mutations in RPL5 or RPL11, and only 1.6% in RPS24. All but three mutations that we report here are new mutations. No mutations were found in RPS14, RPS16, or RPL35A. Remarkably, we observed a higher percentage of somatic malformations in patients with RPL5 and RPL11 mutations. A close association was evident between RPL5 mutations and craniofacial malformations, and between hand malformations and RPL11 mutations. Conclusions Mutations in four ribosomal proteins account for around 50% of all cases of Diamond-Blackfan anemia in Italian patients. Genotype-phenotype data suggest that mutation screening should begin with RPL5 and RPL11 in patients with Diamond-Blackfan anemia with malformations.

Familial tumoral calcinosis and testicular microlithiasis associated with a new mutation of GALNT3 in a white family.

Background: Familial tumoral calcinosis (FTC) is a rare autosomal recessive disease characterised by the development of multiple calcified masses in periarticular soft tissues; GALNT3 gene mutations have recently been described in an African American and in a Druse Arab family with FTC. Objective: To report the clinical and histological features caused by a new GALNT3 mutation in a white family. Results: Homozygosity for the nonsense mutation Lys463X was found in both affected siblings, who displayed a classic phenotype, the male also having testicular microlithiasis. He is the first subject described with testicular microlithiasis in FTC. Conclusions: The high testicular expression of GALNT3 suggests that the gene alteration could act locally by causing deposition of calcium, and the testis may be an underestimated site of calcification in FTC. Autoimmune diseases are present in several members of the family. Although immune disorders have been described in FTC, autoimmunity does not segregate with the GALNT3 mutation in this family.

Loss of GATA-1 Full Length as a Cause of Diamond–Blackfan Anemia Phenotype

Mutations in the hematopoietic transcription factor GATA‐1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full‐length isoform of GATA‐1 and lead to the production of a shortened isoform, GATA‐1s. These mutations have been found in patients with Diamond–Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA‐1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full‐length GATA‐1 isoform. Pediatr Blood Cancer 2014;61:1319–1321.

Rapp-Hodgkin and AEC syndromes due to a new frameshift mutation in the TP63 gene

Increases in the number of allelic malformation syndromes (due to mutations in a single gene) have led to their classification according to their pathogenesis rather than their clinical specific phenotype. TP63 mutations have been identified in several such syndromes characterised by autosomal dominant transmission and various combinations of ectodermal dysplasia, limb malformations, and orofacial clefting. The TP63 gene is a TP53 homologue,1–8 part of a family composed of only three members. The third member (TP73) is more similar to TP63 than to TP53 in both structure and function.9–13 Like p53, p63 has a transactivating (TA), a DNA binding (DB), and a polymerisation domain; it exerts p53-like activities in various contexts, such as binding canonical p53 sites, transactivating p53 target genes, and inducing apoptosis.1,2 Unlike TP53, which expresses one major transcript, TP63 contains four separate transcription initiation sites that direct expression of two fundamentally different isotypes that retain (TA products) or lack (ΔN products) the TA domain.14 Alternative splicing generates additional complexity at the C terminus. ΔN isoforms lack TA activity and may also suppress the TA isoforms, either by simple competition for the DNA target sites or by acting as dominant negatives through oligomerisation. By contrast to p53, the C terminus in p63 is longer and contains a SAM domain and a TID (transactivation inhibitory domain). SAM domains are involved in protein-protein interactions and probably have regulatory functions in p63,15–17 since its TA-α isoform shows a lower TA activity than the γ form, which lacks the SAM but retains the TA domain. The TID has been mapped within the α tail downstream to the SAM domain.14 The differences at the C terminus identify three transcripts which have different properties and functions: α, β, and γ isoforms. The α isoforms have …

High frequency of ribosomal protein gene deletions in Italian Diamond-Blackfan anemia patients detected by multiplex ligation-dependent probe amplification assay.

Diamond-Blackfan anemia is an autosomal dominant disease due to mutations in nine ribosomal protein encoding genes. Because most mutations are loss of function and detected by direct sequencing of coding exons, we reasoned that part of the approximately 50% mutation negative patients may have carried a copy number variant of ribosomal protein genes. As a proof of concept, we designed a multiplex ligation-dependent probe amplification assay targeted to screen the six genes that are most frequently mutated in Diamond-Blackfan anemia patients: RPS17, RPS19, RPS26, RPL5, RPL11, and RPL35A. Using this assay we showed that deletions represent approximately 20% of all mutations. The combination of sequencing and multiplex ligation-dependent probe amplification analysis of these six genes allows the genetic characterization of approximately 65% of patients, showing that Diamond-Blackfan anemia is indisputably a ribosomopathy.

A new database for ribosomal protein genes which are mutated in Diamond‐Blackfan Anemia

Mutations in ribosomal proteins RPS19, RPS24 and RPS17 have been reported in Diamond‐Blackfan Anemia (DBA), an autosomal dominant disease characterised by pure red cell aplasia. DBA is the prototype of ribosomapathies: a protein synthesis defect in a tissue with a high cellular turnover is considered the cause of the erythroid progenitor failure. We have created the Diamond‐Blackfan Anemia mutation database to curate and record DBA gene mutations, together with their functional consequences and clinical phenotypes. This locus‐specific resource is open to future submissions and is available online (http://www.dbagenes.unito.it). It is founded on the Leiden Open (source) Variation Database (LOVD) system and includes data from sequence and structure analysis tools, genomic database resources and published reports. It lists all identified variants and background genomic information. Phenotypic data are accessed by selecting a particular mutation. The database includes 219 unique variants of which 86 are disease‐causing mutations. The database will be supplemented with other DBA genes as soon as they are reported and their mutations are identified and it should be of assistance to clinicians and investigators involved in DBA research and care.

HDR Syndrome : A Novel de novo Mutation in GATA3 Gene

Human GATA3 haploinsufficiency leads to HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome. The development of a specific subset of organs in which this transcription factor is expressed appears exquisitely sensitive to gene dosage. We report on a 14‐year‐old patient with symptomatic hypoparathyroidism, sensorineural bilateral deafness, unilateral renal dysplasia, bilateral palpebral ptosis, and horizontal nystagmus. Fundoscopy displayed symmetrical pseudopapilledema, and brain CT scan revealed basal ganglia calcifications. FISH analysis did not disclose any microdeletion in the 22q11.2 or 10p14 regions. GATA3 mutation analysis identified a heterozygous deletion of GG nucleotides at codon 36 and 37 (c.108_109delGG) in exon 2 causing a frameshift with a premature stop codon after a new 15‐aminoacid sequence. Restriction endonuclease analysis performed in parents was negative. Our patient carries a novel “de novo” GATA3 mutation, providing further evidence that HDR syndrome is caused by haploinsufficiency of GATA3, which may be responsible for a complex neurologic picture besides the known triad.

Multiplex ligation-dependent probe amplification enhances molecular diagnosis of Diamond-Blackfan anemia due to RPS19 deficiency

Diamond-Blackfan anemia (DBA,#MIM105650) is a rare congenital pure red cell aplasia characterized by nor-mochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors. Defects in the RPS19 gene on chromosome 19q13.2 are the main

R298Q mutation of p63 gene in autosomal dominant ectodermal dysplasia associated with arrhythmogenic right ventricular cardiomyopathy

Mutations in the p63 gene have been identified in five types of syndromic ectodermal dysplasias (EDs) with overlapping phenotypes: Ectrodactyly-Ectodermal dysplasia-Clefting (EEC syndrome, MIM 604292), Ankyloblepharon-Ectodermal dysplasia-Clefting (AEC syndrome, MIM 106260) [3], Acro-Dermato-Ungueal-Lacrimal-Tooth (ADULT syndrome, MIM 103285), Rapp-Hodgkin (RHS syndrome, MIM 129400) and Limb-Mammary (LMS syndrome, MIM 603543) [2]. In all those conditions congenital heart defects have been only occasionally found and to date, arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) has never been observed in patients affected by p63-related ectodermal dysplasia [9]. Here we describe for the first time this association.