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LethalALAS2mutation in males X-linked sideroblastic anaemia

2016; Wiley; Volume: 178; Issue: 4 Linguagem: Inglês

10.1111/bjh.14164

1365-2141

Christian Rosé, Isabelle Callebaut, Laurent Pascal, Claire Oudin, Martine Fournier, Laurent Gouya, Anne Lambilliotte, Caroline Kannengiesser,

Neonatal Health and Biochemistry

X-linked Sideroblastic Anaemia (XLSA, Mendelian Inheritance in Man reference 300751), the most common type of congenital sideroblastic anaemia (CSA), results from germline mutations in the erythroid-specific form of the 5-aminolevulinate synthase gene (ALAS2). ALAS2 is the first enzyme of the haem biosynthesis pathway and is essential to support haemoglobin synthesis. Affected males typically exhibit microcytic anaemia and iron deposits in the mitochondria of erythroid precursors (ring sideroblasts). Anaemia is generally mild and patients are rarely transfusion dependent. However patients could exhibit, with time, severe parenchymal iron overload due to intestinal hyper absorption of iron (Cazzola & Malcovati, 2015). Most of the ALAS2 mutations are missense mutations with partial loss-of-function alleles; mutations in a GATA transcription factor-binding in intron 1 of the ALAS2 gene have also been reported (Campagna et al, 2014). However penetrance and phenotypic expression of XLSA is highly variable, even in a same family(Cortesão et al, 2004; Aguiar et al, 2014). Up to one-third of female cases may be affected, mainly by skewed X-chromosome inactivation which increases with age (Ducamp et al, 2011); one case of macrocytic anaemia in a female with dominant mutation in ALAS2 without skewed X inactivation has also been reported to alter erythropoiesis through cell non-autonomous effects (Sankaran et al, 2015). We report for the first time in a very large, four-generation pedigree a new missense mutation in the ALAS2 gene inducing a Male Lethal X-linked syndrome ascertained through an adult heterozygous female with highly skewed X inactivation with a mild form of macrocytic CSA and an excessive number of miscarriages. The proposita of this non-consanguineous family (Fig 1A; Individual III.9) was a female of European ancestry. She exhibited an unexplained congenital, non-regenerative, macrocytic, moderate anaemia, (first assessment at 6 years old). Red blood cell (RBC) transfusions were required only twice during her pregnancy. She was diagnosed with CSA when aged 23 years. A complete blood count showed a non-regenerative macrocytic [mean cell volume (MCV) 107 fl] anaemia (Hb: 107 g/l)that was not dimorphic, with red cell distribution width (RDW) -SD 52·5 fl [normal range (N): 37–50; significant anisocytosis if > 65], RDW-CV: 13·6% (N:11–14%), microcytes 1·9% (N: <6%), macrocytes 13·8% (N: <6%). Bone marrow aspiration showed 38% ring sideroblasts (Fig 1B) with massive dyserythropoiesis without different pattern repartition in term of apoptosis or dyserythropoiesis. The serum ferritin level was 224 μg/l (N: 12–150) and transferrin saturation was 90%. The liver iron concentration by magnetic resonance imaging was 298 μmol/g dry weight (N < 36 μmol/g dry weight). A rare heterozygous deleterious missense mutation in exon 5 of ALAS2 (NM_000032) c.622G>T, p.Val208Phe (Fig 1 C) was found affecting a totally conserved amino acid (in silico prediction Polyphen score =1). A constitutive highly skewed X-chromosome inactivation was demonstrated by human androgen receptor gene (HUMARA technique (85–15%) as previously reported in affected females with XLSA. The propositas' daughter and mother were unaffected, with a normal blood count; they were also carriers of the same mutation but did not have skewed X-chromosome inactivation. A close inspection of the pedigree showed a large female predominance (22 females/7 males) over four generations (female/male ratio 3·1). No affected males were identified in the pedigree. A large number of miscarriages (18 over the four generations) were found. Molecular analysis of ALAS2 gene of the pedigree showed that the miscarriages occurred only in females carrying the ALAS2 mutation (Fig 1A). In living male members of the family available for genetic testing (Individuals III. 8 and III.12), the search of ALAS2 mutation was negative. Adding the number of miscarriages (highly suspected to be males) to the number of males alive, the new female/male ratio over 4 generations becomes close to 1 (25/22). Extensive investigation into the causes of recurrent miscarriages was negative in Individual II.7. No chromosomal or molecular analysis has been performed on the products of conception. In order to understand the molecular basis of the missense mutation impact, we built a model of the 3D structure of human ALAS2 catalytic core, based on experimental 3D structure of R. capsulatus ALAS, which shares 49% sequence identity with human ALAS2, with only a few insertions-deletions outside the conserved regular secondary structures (Fig 2A) (Astner et al, 2005). The analysis of this 3D model predicted a strong impact of the mutation on the 3D structure, by disturbing the fold of the monomer as well as the stability of the dimeric architecture (Fig 2B). These modelling data confirm the extensive familial study showing excessive miscarriages, and highly suggests an X-linked disorder with prenatal male lethality. The unusual phenotype of this XLSA (macrocytic, absence of dimorphic RBC population) in this female may be explained by the severity of the mutation, leading to intramedullary apoptosis of mutated erythroid precursors and allowing only wild-type erythroid cells to reach the circulation. Our case highlights the heterogeneity of XLSA and the non-redundant crucial role of the erythroid-specific form of delta-aminolevulinate synthase in fetal haematopoiesis. Mutagenesis studies are required to fully elucidate the role of this new mutant, which is predicted to affect a critical amino acid, based on 3D modelling. Moreover a molecular study has shown that, in affected heterozygotes females from 1 independent family, only wild type cells persisted during erythroid differentiation, suggesting a very deleterious effect of some variants (Sankaran et al, 2015). Miscarriages could have occurred in other previous small published families with females cases in which females are more numerous than males (Cotter et al, 1995; Cortesão et al, 2004; Ducamp et al, 2011). Moreover, a remarkable degree of heterogeneity in the X chromosome inactivation is frequently reported in XLSA (Cazzola & Malcovati, 2015), as it has also previously been described in X-linked dominant lethal disorders (Franco & Ballabio, 2006). Our family study clearly indicates that investigation of excessive miscarriages in the pedigree of XLSA- affected females should be considered, and that the spectrum of the disease should be extended to include male lethal X-linked disorders. CR, LP, AL followed the patients and collected the clinical data; ID performed ALAS2 3D structure modelling; CO, LG, CK performed genetic and molecular analysis; MF performed bone marrow cytological analysis; CR, ID, CK wrote the report. All authors contributed to data analysis and manuscript review. The authors have no conflict of interest to disclose.