Absence of acquired copy number neutral loss of heterozygosity (CN-LOH) of chromosome 7 in a series of 10 patients with Shwachman-Diamond syndrome.

We report that acquired copy number neutral loss of heterozygosity (CN-LOH) of chromosome 7 was not identified in a series of 10 patients with Shwachman–Diamond syndrome (SDS). Shwachman–Diamond syndrome (Online Mendelian Inheritance in Man reference 260400) is a rare autosomal recessive disease first reported in 1964 (Bodian et al,1964; Shwachman et al,1964) and characterized by exocrine pancreatic insufficiency, skeletal abnormalities and bone marrow (BM) dysfunction with an increased risk to develop myelodysplastic syndrome and/or acute myeloid leukaemia (MDS/ AML). Almost 90% of SDS cases are caused by two common mutations, c. [183_184TA>CT] and c.[258 + 2T>C], in exon 2 of the SBDS gene, localized on chromosome 7. Clonal chromosome anomalies are often found in the BM of SDS patients; the most frequent are an isochromosome for long arms of chromosome 7, i(7)(q10) and an interstitial deletion of long arms of chromosome 20, del(20)(q11) (Maserati et al, 2009). An earlier study analysed BM DNA of eight cases with the i(7)(q10) and demonstrated that all of them carried a double dose of the c.[258 + 2T>C] (Minelli et al, 2009). This result suggested that, as the c.[258 + 2T>C] mutation still allows the production of some amount of normal protein (Austin et al, 2005), this could contribute to the low incidence of MDS/AML observed in this subset of SDS patients. Recently, Parikh et al (2012) described acquired CN-LOH for most of 7q in a patient with SDS, also showing that the clone of BM cells with CN-LOH contained two copies of the gene with the c.[258 + 2T>C] mutation. The presence of the CN-LOH in BM cells mimics the presence of i(7)(q10): both mechanisms produce a duplication of the c.[258 + 2T>C] mutation, together with the probable associated positive effects. Parikh et al (2012) also outlined that the genetic variation of the CN-LOH could provide an explanation for clonal expansion of the affected haematopoietic progenitor cell. Consequently, we investigated if, to provide the cell with an extra copy of the c.[258 + 2T>C] mutation, this could be a common mechanism in addition to the typical i(7)(q10) frequently observed in BM from SDS patients. We collected BM samples from 10 SDS patients in whom the cytogenetic analysis demonstrated a normal karyotype (eight cases) or the presence of the 20q deletion (two cases). The genotypes were c.[258 + 2T>C]/[183_184TA>CT] in seven cases and c.[258 + 2T>C]/[others] in three cases. Morphological analysis of the BM aspirate, available for six patients, showed normal or hypoplastic cellularity in three, and normal or hyperplastic cellularity with minimal decrease of erythropoiesis, megakaryopoiesis and myelopoiesis in the remaining three. These data are in keeping with the karyotype-phenotype correlation discussed by Pressato et al (2012). Microsatellite analysis, performed as reported in Minelli et al (2009), demonstrated a normal dosage between paternal and maternal alleles, excluding the presence of uniparental disomy related to chromosome 7, in all BM samples examined (Table I and Fig 1A). The method is able to identify the presence of the chromosomal anomaly if it is present in at least 20% of cells (Minelli et al, 2009). To verify the reliability of the method, we sequenced exon 2 of the SBDS gene from the BM of two SDS patients with 50–70% cells with i(7)(q10) and, as expected, found that the mutant peak (C) was higher than the wild-type peak (T) (Fig 1B). Exon 2 of the SBDS gene was then sequenced using BM and peripheral blood samples and, in all cases analysed, the mutant peak (C) and the wild-type peak (T) for the c.[258 + 2T>C] mutation were exactly the same height in both samples (Fig 1C) whereas Parikh et al (2012) observed an higher peak for the mutated allele when BM and fibroblasts were compared. Thus, no evidence for CN-LOH was found in any of the patients studied. If the hypothesis that an increased level of the c.[258 + 2T>C] mutation on a SDS background (c.[183_184TA>CT]/c.[258 + 2T>C]) provides a selective advantage, we would expect to identify cases in whom a clone showing a (partial) loss of the c.[183_184TA>CT] or an homozygous c.[258 + 2T>C] is present.