Albertine Donker

Chapter 6 206 PATIENTS AND METHODS Patients We reviewed clinical and molecular data of 15 patients (14 male and one female) diagnosed with XLSA in the Netherlands in 2011 and 2012. These patients were diagnosed with sideroblastic anemia at the University Medical Center Utrecht, Utrecht and the Radboud University Medical Center, Nijmegen, the Netherlands. We reviewed age at presentation, biochemical and clinical features, treatment regimens and type of ALAS2 mutations. Genotyping Genotyping was performed by PCR and DNA sequence analysis of the full coding part of the ALAS2 gene. The pathogenicity of a mutation was assessed by review of the literature, association of the mutation with the phenotype in a family and bio-informatic tools. The latter tools were used to complement the genetic studies in case of a not previously reported mutation. SIFT (=Sorting Intolerant from Tolerant) and PolyPhen (Polymorphism Phenotyping) and HOPE (Have (y)Our Protein Explained) provide an in-silico prediction of the functional consequences of missense mutations. 14-17 Search for a founder effect was done in 7 of the 10 families with the p.Arg452His mutation by genotyping the Short Tandem Repeats (STR’s) DXS1044, DXS8032, DXS991 and DXS1190 close to the ALAS2 gene by PCR using fluorescent primers. PCR products were pooled and analyzed on an ABI 3730 DNA sequences.