Marie-José Grégoire

Clinical phenotype of germline RUNX1 haploinsufficiency: from point mutations to large genomic deletions

Germline RUNX1 mutations result in a rare autosomal dominant condition characterized by qualitative and quantitative platelet defects and predisposition to the development of myeloid malignancies (familial platelet disorder with propensity to acute myeloid leukaemia, FPD/AML). Only 13 pedigrees have previously been described so far. We report on two novel germline RUNX1 mutations: (1) an out-of-frame 8 bp heterozygous deletion (c.442_449del) in an FPD/AML pedigree and (2) a de novo 3.5 Mb deletion in the 21q22.11.21q22.12 region encompassing the RUNX1 gene in a mentally retarded female patient with short stature and thrombocytopenia. Interestingly, a similar de novo submicroscopic deletion has been recently reported in the literature in a mentally retarded patient. Mental retardation is one of the most common disorders and primary causes of thrombocytopenia are rare. When occurring together, these features should prompt to test for 21q22 deletion for comprehensive genetic counselling and clinical management.

The 2q37-deletion syndrome: an update of the clinical spectrum including overweight, brachydactyly and behavioural features in 14 new patients.

The 2q37 locus is one of the most commonly deleted subtelomeric regions. Such a deletion has been identified in >100 patients by telomeric fluorescence in situ hybridization (FISH) analysis and, less frequently, by array-based comparative genomic hybridization (array-CGH). A recognizable ‘2q37-deletion syndrome’ or Albright’s hereditary osteodystrophy-like syndrome has been previously described. To better map the deletion and further refine this deletional syndrome, we formed a collaboration with the Association of French Language Cytogeneticists to collect 14 new intellectually deficient patients with a distal or interstitial 2q37 deletion characterized by FISH and array-CGH. Patients exhibited facial dysmorphism (13/14) and brachydactyly (10/14), associated with behavioural problems, autism or autism spectrum disorders of varying severity and overweight or obesity. The deletions in these 14 new patients measured from 2.6 to 8.8 Mb. Although the major role of HDAC4 has been demonstrated, the phenotypic involvement of several other genes in the deleted regions is unknown. We further refined the genotype–phenotype correlation for the 2q37 deletion. To do this, we examined the smallest overlapping deleted region for candidate genes for skeletal malformations (facial dysmorphism and brachydactyly), overweight, behavioural problems and seizures, using clinical data, a review of the literature, and the Manteia database. Among the candidate genes identified, we focus on the roles of PRLH, PER2, TWIST2, CAPN10, KIF1A, FARP2, D2HGDH and PDCD1.

Aberrant GRIA3 transcripts with multi-exon duplications in a family with X-linked mental retardation.

Investigation of chromosomal rearrangements in patients with mental retardation (MR) is particularly informative in the search for novel genes involved in MR. We report on a family with a genomic duplication at Xq25 identified by oligo array‐CGH. Further characterization showed a partial tandem duplication of GRIA3 extending from exon 1 to exon 12. This duplication is present in two brothers with MR and on one allele in their sister with normal phenotype and completely skewed X‐chromosome inactivation. The duplication is inherited from the mother, whose cognitive level is low and X‐chromosome inactivation is random. This is the second family with partial duplication of GRIA3 associated with MR. GRIA3 expression studies in our case demonstrated a new mechanism for GRIA3 dysfunction with the presence of aberrant GRIA3 transcripts carrying multi‐exon duplications leading to a frameshift. Our study gives additional support to the implication of GRIA3 in X‐linked MR.

Clinical and molecular characterization of a large family with an interstitial 15q11q13 duplication

The clinical significance of an interstitial duplication of chromosome 15q11q13 is still not well documented. This abnormality has been associated with autistic spectrum disorders (ASD) and varying degrees of mental retardation. The clinical variability appears to be influenced by the parental origin of the duplication. We present here the clinical evaluation and psychological assessment of the largest reported family with 12 carriers on three generations. Patients exhibit mental retardation, motor and visuo‐motor skills impairments and adaptive functioning deficit without formal diagnosis of autism. There appeared to be evidence in the family of reduced penetrance in duplication of paternal origin. This familial 15q11q13 duplication was precisely investigated by cytogenetic and molecular techniques including fluorescence in situ hybridization (FISH), PCR analysis of microsatellite markers, array‐comparative genomic hybridization analysis (Array‐CGH) and semi‐quantitative methylation‐sensitive PCR. Results showed an inherited 15q11q13 duplication of maternal origin in 10 patients and of paternal origin in the remaining two. The size of the duplicated area was around 6 Mb with breakpoints in accordance with those previously reported. This report extends the clinical spectrum of the 15q11q13 duplication, and we recommend the investigation of 15q11q13 duplication not only in subjects with autistic spectrum disorder but also in patients with low normal intelligence and dyspraxia.

Identification of an acute basophilic leukaemia carrying a rare e6a2 BCR-ABL transcript.

ripheral blood analysis showed moderate anaemia (Hb 10 g/dl), thrombocytopenia (platelet count 51 ! 10 9 /l) and hyperleucocytosis (WBC 80.8 ! 10 9 /l), which included 8% myeloid cells and 30% blasts. Of these, 24% had coarse basophilic granules and 6% were undifferentiated blasts. There were 8% basophilic precursors and 5% mature basophils. Bone marrow aspirate revealed a cellular marrow with 33% blasts including 28% with coarse basophilic granules and 5% undifferentiated blasts associated with 12% basophilic precursors and 4% mature basophils ( fig. 1 a). Auer rods were not seen. The blasts were negative for myeloperoxidaxe and nonspecific esterase. Basophilic granules in the blasts and basophils exhibited metachromasia with toluidine blue. Immunophenotyping with four-colour flow cytometry was performed on bone marrow. Blasts were gated on their dim CD45 and low sidescatter characteristics. They expressed a myeloid phenotype since they were positive for CD13, CD33, CD11c and CD203c, a specific basophilic marker. Blasts were negative for early haematopoietic markers (CD34, HLA-DR, TdT), for B and T lymphoid markers (CD10, CD19, CD20, CD22, CD2, CD3, CD5) and for other myeloid markers (CD14, CD15, CD65, CD117, MPO). Cytogenetic analysis, performed on 24and 48-hour bone marrow cultures, The t(9; 22)(q34;q11) translocation has been implicated as the causative factor in greater than 95% of chronic myelogenous leukaemia (CML), in 25–30% of adult and 2–10% of childhood acute lymphoblastic leukaemia (ALL) and in rare acute myelogenous leukaemia (AML). This translocation generates a fusion gene between the 5 part of the BCR gene and the 3 part of the c-ABL gene. In CML, the breakpoint in BCR usually falls in the major breakpoint cluster region (M-bcr) with junctions e13a2, e14a2 and less frequently e13a3, e14a3, as in two thirds of ALL. In rare cases of CML and other ALL cases, it is located in minor breakpoint (m-bcr) with junctions e1a2 and rarely e1a3. More recently, a third bcr region called micro-bcr was detected with juxtaposition of exon 19 to ABL (e19a2) [1] . In this report we describe a new case of BCR-ABL fusion with chromosome 22 breakpoint unusually located in BCR intron 6 resulting in e6a2 junction. Only six cases have been reported to our knowledge [2–7] , and it is the first one associated with acute basophilic leukaemia features. A 71-year-old male was referred to our care unit because of a bilateral pneumopathy. On clinical examination, there was no hepatosplenomegaly or cutaneous lesions or symptoms related to hyperhistaminaemia. PeReceived: February 8, 2006 Accepted after revision: February 14, 2006

Genotype–phenotype correlations to aid in the prognosis of individuals with uncommon 20q13.33 subtelomere deletions: a collaborative study on behalf of the ‘association des Cytogénéticiens de langue Française’

The identification of subtelomeric rearrangements as a cause of mental retardation has made a considerable contribution to diagnosing patients with mental retardation. It is remarkable that for certain subtelomeric regions, deletions have hardly ever been reported so far. All the laboratories from the ‘Association des Cytogénéticiens de Langue Française’ were surveyed for cases where an abnormality of the subtelomere FISH analysis had been ascertained. Among 1511 cases referred owing to unexplained mental retardation, 115 (7.6%) patients showed a clinically significant subtelomeric abnormality. We report the clinical features and the molecular cytogenetic delineation of isolated de novo deletions on 20q13.33 in two cases. Detailed mapping was performed by micro-array CGH in one patient and confirmed by FISH in the two patients. We compare our data with the only three patients reported in the literature. Both patients shared a deleted region of approximately 1.33 Mb including 40 genes, with a 324 kb difference between the two patients. Haploinsufficiency for CHRNA4 and ARFGAP1 may have contributed towards a severe phenotype. In addition, the data in all patients suggest that haploinsufficiency for SOX18 may not cause the hypotrichosis–lymphedema–telangiectasia syndrome, or causes milder disease. Our study gives important information by defining the size of imbalance and better predicting the phenotype. Two clinically distinct phenotypes may be drawn, a mild mental retardation or a more complex and severe phenotype, according to the presence or absence of the CHRNA4 and ARFGAP1 genes respectively.

Characterization of mosaic supernumerary ring chromosomes by array-CGH: Segmental aneusomy for proximal 4q in a child with tall stature and obesity

We report on a 6‐year‐old girl with developmental delay, tall stature, and obesity. G‐banded chromosome analysis revealed mosaicism for one to three small de novo rings in 82% of peripheral lymphocytes. Fluorescence in situ hybridization (FISH) studies and metaphase comparative genomic hybridization (CGH) demonstrated that the rings were derived from 4q10‐4q13. A higher resolution investigation was initiated using array‐CGH analysis and revealed a gain of 11 adjacent clones spanning a 16 Mb region at 4q11‐q13.2 and including the insulin‐like growth factor binding protein 7 (IGFBP7) gene. This finding suggests that postnatal overgrowth observed in our patient might be related to a dosage effect of the IGFBP7 gene.

Molecular characterization of a monosomy 1p36 presenting as an Aicardi syndrome phenocopy

Monosomy 1p36 is the most frequent terminal deletion known in Humans. Typical craniofacial features, developmental delay/mental retardation, seizures and sensorineural defects characterize 1p36 deletion syndrome. Aicardi syndrome (AIS) is a rare genetic disorder characterized by chorioretinal lacunae, corpus callosum agenesis and infantile spasms responsible for mental retardation. By screening DNA from diagnosed AIS patients with oligonucleotide array‐based comparative genomic hybridization (aCGH), we report a 1p36 monosomy in this study. There were no other deletions or duplications. Regarding clinical criteria, the patient did not have the typical facial appearance commonly described for 1p36 monosomy patients. We showed that this 1p36 monosomy corresponded to combined interstitial and terminal de novo deletions of the chromosome 1 leading to an 11.73 Mb deletion confirmed with qPCR. By microsatellite markers and FISH analyses, we have concluded that this deletion occurred on maternal chromosome 1 during oogenesis. We did find some clinical features shared by the 1p36 monosomy and AIS: infantile spasms, corpus callosum dysgenesis, ophthalmological abnormalities, and skeletal malformations. To date, no relationship between these two phenotypes has been established. We conclude that the monosomy 1p36 should be considered in the differential diagnosis of AIS.

De novo methylation of tumour suppressor genes CDKN2A and CDKN2B is a rare finding in B-cell chronic lymphocytic leukaemia

De novo methylation of the 5′CpG island has been recently reported as an alternative mechanism of inactivation for the tumour suppressor genes CDKN2A and CDKN2B. We examined CDKN2A methylation status at diagnosis in 42 B‐cell chronic lymphocytic leukaemia (CLL) patients, in 19 cases the CDKN2B methylation status was also analysed. No homozygous CDKN2A/2B deletion was detected, but four patients (9%) displayed an aberrant CDKN2A methylation status and only one had hypermethylated CDKN2B. De novo methylation was associated with silencing of gene expression. These results confirm that CDKN2A/2B inactivation by deletion is a rare event in CLL and suggest that aberrant methylation could be an alternative way of inactivation very rarely involved in the development of some CLL.

CD203c+/CD117−, an useful phenotype profile for acute basophilic leukaemia diagnosis in cases of undifferentiated blasts

Acute basophilic leukaemia is a rare acute myeloid leukaemia, in which the primary differentiation is to basophiles. Diagnosis is generally made using classical morphological, cytochemical methods and, in some cases, immunological, cytogenetic and molecular genetic data. Most of the blast cells are morphologically undifferentiated but some of them may contain coarse basophilic granules, recognition of which should be the first step in diagnosis of this disorder. The presence of coarse deep purple granules is highly significant, but varies from case to case. Indeed, some authors reported blasts in which basophilic granules were not seen [1,2]. The myeloperoxydase reaction remains poorly contributive to the diagnosis since it is generally found negative: 14/16, 1/2 and 5/8 cases respectively in Duchayne [1], Giagounidis [3] and Shvidel [2] studies. Moreover, the typical toluidine blue metachromatic reaction is positive in only a proportion of cases, depending on the maturity of the basophile granules. Three of eight cases were found to be negative [2]. This lack of positivity could result from the immaturity of these basophilic blasts. Immunophenotypic studies showed a wide range of variation. A positivity for the panleukocyte antigen CD45, for the immaturity markers (CD34, HLA-DR) and for the myeloid markers CD13, CD33 and CD11b were reported. B and T lymphoid markers were found to be negative [1]. Cytogenetic investigations give heterogeneous and non-specific results. A few cases may present as de novo Philadelphia chromosome positive acute leukaemias, with a t(9;22) (q34;q11). In Shvidel study [2], definitive diagnosis of acute basophilic leukaemia should be made only by electronic microscopy, which demonstrated the presence of basophilic granules in the blast cells. Ultrastructural demonstration of peroxidase in the granules is observed in all cases. The authors suggested that this exploration should be systematically included in diagnosis criteria of poorly differentiated acute leukaemias. We report a case of a 71-year-old man that was referred to our care unit for a bilateral pneumopathy. On clinical examination, there was no hepatosplenomeagly or cutaneous lesions or symptoms related to hyperhistaminemia. Peripheral blood analysis showed moderate anaemia (Hb 10 g/dL), thrombocytopenia (platelet count 51610/L) and hyperleucocytosis (WBC 80.8610/L), which included 8% myeloid cells and 30% blasts. Of these, 24% had coarse basophilic granules and 6% were undifferentiated blasts. There were 8% basophilic precursors and 5% mature basophiles. Bone marrow aspirate revealed a cellular marrow with 33% blasts including 28% with coarse basophilic granules and 5% undifferentiated blasts associated with 12% basophilic precursors and 4% mature basophiles (Figure 1a). Auer rods were not seen. The blasts were negative for myeloperoxidaxe and non-specific esterase. Basophilic granules in the blasts and basophiles exhibited metachromasia with toluidine blue. Immunophenotyping with four-colour flow cytometry was performed on bone marrow. Blasts were