Phenotype / Genotype Correlations in Diamond-Blackfan Anaemia – An Update From the Czech National DBA Registry
Authors: Pospisilova Dagmar , Cmejlova Jana , Stary Jan , Cerna Zdena , Hak Jiri , Cmejla Radek , Ludikova Barbora 
Published: October 2019

Abstract
Abstract 5269

Introduction
Diamond-Blackfan anaemia (DBA) is a rare congenital red cell aplasia that is also associated with various physical anomalies in 40% of patients. Haploinsufficiency of ribosomal proteins (RPs) production due to various mutations in RPs is believed to be the cause of DBA. However, the precise mechanism of erythroid failure and development of anomalies remains under debate. Here we report a summary of clinical and laboratory data from the Czech National DBA Registry.

Patients and Methods
The Czech DBA registry has been created in the period 1991–2011. All patients were examined by experienced haematologists and detailed history was obtained. The following analyses were done: bone marrow analysis, eADA levels and clonogenic assays as previously published. PCR and direct sequencing was used to identify mutations in the genes coding for the following 21 RPs: RPS2, RPS3, RPS3a, RPS10, RPS12, RPS13, RPS14, RPS16, RPS17, RPS19, RPS24, RPS25, RPS30, RPL5, RPL11, RPL13, RPL23, RPL26, RPL27, RPL35a and RPL36.

Results
The Czech DBA Registry currently comprises 39 patients (14 males and 25 females; 1:1.79 ratio) aged 6 months-53 years from 34 families. Seventeen (28.8 %) patients were born small for gestational age (SGA), which is significantly higher in comparison with the population of Czech healthy newborns (p>0,001). In 27 (69.2%) patients, one or more anomalies were found (thumb anomalies, high-arched palate, craniofacial dysmorphism, Klippel-Feil syndrome, Sprengel`s deformity, neck, heart and kidney anomalies, microcephaly, micropthalmia). Nineteen (48.7%) patients have short stature. Only 6 (15%) patients have neither anomalies nor short stature. Two patients (5.1%) have developed malignancy. The first one died at the age of 5 due to AML, the second patient with an RPL11 mutation developed non-Hodgkin lymphoma at the age of 36. eADA levels were increased in 16/18 (88.8%) of non-transfused patients. Eighteen (46.2%) patients are transfusion dependent, while 10 (24.6%) are in remission and 11(28.2%) are on steroid treatment. So far, 23 different heterozygous mutations in five different RPs – RPS17, RPS19, RPS26, RPL5, and RPL11 – have been identified in 28 patients (71.8%) from 23 families (67.6%). Most mutations were found in the RPS19 gene – 10 patients (25.6%) from 8 families (23.5%), followed by the RPL5 gene (8 patients (20.5%) from 6 families (17.6%)); the RPS26 gene (5 patients (12.8%) from 5 families (14.7%)); the RPL11 gene (4 patients (10.3%) from 3 families (8.8%)); and RPS17 (1 patient (2.6%) from 1 family (2.9%).
We identified three new mutations in the RPS19 gene (c.58G>C, p.Ala20Pro; c.195C>G, p.Tyr65X; and c.356dupG, p.Gly120ArgfsX34), one new mutation in the RPS26 gene (c.6_9delAAAG, p.Lys4GlufsX40), and one familial mutation in the RPL11 gene (c.281T>G, p.Leu94X).
The comparison of the group of patients with RPS19 mutations (n=10) with the group of patients with RPL5 and 11 mutations (n=12) showed two significant differences. Firstly, 11/12 (92%) patients with RPL5 or RPL11 mutations were born SGA, secondly, all patients with an RPL5 or RPL11 mutation have a thumb defect with one or more other anomalies, while in the RPS19-mutated group the frequency of both markers was lower (30%). RPL5 and RPL11 mutations seem to have more profound impact on fetal development than mutations in RPS19. Patients with an RPS26 mutation have no thumb anomalies, but they show other skeletal (ribs and neck) anomalies.

Conclusions
The incidence of DBA in the Czech Republic is calculated to be 8.1/1 million live births. We observed a higher frequency of associated anomalies (70%) than was previously reported. Mutations in RPs of the small ribosomal subunit were found in 14 families (60.9%), while 9 mutations in proteins of the large ribosomal subunit represented 39.1%, together amounting to 72% of resolved DBA cases in the Czech Republic. Four new mutations have not been described yet. We found significant genotype-phenotype correlations. National registries are therefore the important tool for better understanding of several aspects of the disease.
The work was supported by grants 00023736 (RC, JC) and NT 11059 (DP) from the Ministry of Health, and MSM 6198959205 from the Ministry of Education, Czech Republic.

Disclosures:
No relevant conflicts of interest to declare.