Bozana Zlateska

Improving diagnostic precision, care and syndrome definitions using comprehensive next-generation sequencing for the inherited bone marrow failure syndromes

Background Phenotypic overlap among the inherited bone marrow failure syndromes (IBMFSs) frequently limits the ability to establish a diagnosis based solely on clinical features. >70 IBMFS genes have been identified, which often renders genetic testing prolonged and costly. Since correct diagnosis, treatment and cancer surveillance often depend on identifying the mutated gene, strategies that enable timely genotyping are essential. Methods To overcome these challenges, we developed a next-generation sequencing assay to analyse a panel of 72 known IBMFS genes. Cases fulfilling the clinical diagnostic criteria of an IBMFS but without identified causal genotypes were included. Results The assay was validated by detecting 52 variants previously found by Sanger sequencing. A total of 158 patients with unknown mutations were studied. Of 75 patients with known IBMFS categories (eg, Fanconi anaemia), 59% had causal mutations. Among 83 patients with unclassified IBMFSs, we found causal mutations and established the diagnosis in 18% of the patients. The assay detected mutant genes that had not previously been reported to be associated with the patient phenotypes. In other cases, the assay led to amendments of diagnoses. In 20% of genotype cases, the results indicated a cancer surveillance programme. Conclusions The novel assay is efficient, accurate and has a major impact on patient care.

Biallelic Mutations in DNAJC21 Cause Shwachman-Diamond Syndrome

Program in Genetics and Genome Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada; Institute of Medical Science, University of Toronto, Toronto, ON, Canada; Marrow Failure and Myelodysplasia Program, Division of Haematology/Oncology, Department of Paediatrics, The Hospital for Sick Children, Toronto, ON, Canada; Division of Haematology Oncology, Children’s Hospital of Western Ontario, London, ON, Canada; Division of Clinical and Metabolic Genetics, Department of Ophthalmology and Vision Sciences, and Division of Gastroenterology and Nutrition, The

Combined de-novo mutation and non-random X-chromosome inactivation causing Wiskott-Aldrich syndrome in a female with thrombocytopenia

ObjectiveDisorders linked to mutations in the X chromosomes typically affect males. The aim of the study is to decipher the mechanism of disease expression in a female patient with a heterozygous mutation on the X-chromosome.Patients and MethodsClinical data was extracted from the Canadian Inherited Marrow Failure Registry. Genomic ribonucleic acid (DNA) and complementary DNA (cDNA) underwent Sanger sequencing. Protein analysis was performed by flow cytometry. X-inactivation patterns were analyzed by evaluating the DNA methylation status and cDNA clonal expression of several genes on the X-chromosome. SNP array was used for molecular karyotyping of the X-chromosome.ResultsA female with thrombocytopenia, eczema and mild T-lymphocyte abnormalities with extensive negative diagnostic testing, was suspected to have Wiskott-Aldrich syndrome (WAS)/X-linked thrombocytopenia. Although the girl had a mutation (c.397G > A, p.E133K) in only one allele, she was found to have an extremely skewed X-inactivation pattern and no expression of the WAS protein. Family studies using DNA methylation analysis and cDNA clonal expression of several genes on the X-chromosome demonstrated that the patient developed de-novo non-random inactivation of the X-chromosome that does not carry the mutation. Genome-wide high-density molecular karyotyping excluded deletions and amplifications as a cause for the non-random inactivation of one X-chromosome.ConclusionsOur study emphasizes the need to test selected female patients with complete or incomplete disease expression for X-linked disorders even in the absence of a family history.

Analysis of Decisional Conflict Among Parents Who Consent to Hypospadias Repair: Single Institution Prospective Study of 100 Couples

PURPOSE Although obtaining informed consent for distal hypospadias repair is common practice, little is known about the uncertainty or conflict between consenting parents faced with this decision. We systematically evaluated decisional conflict between parents who elected to have their child undergo hypospadias surgery. MATERIALS AND METHODS A total of 100 couples who were counseled about treatment options agreed to participate. Using a validated questionnaire, the Decisional Conflict Scale, we prospectively collected data on decisional conflict demographics, preference for circumcision, education level and prior knowledge about hypospadias. RESULTS All parents elected surgical repair. Evidence of decisional conflict was encountered in 28% of participants (score less than 25 in 72%, 25 to 37.5 in 23.5%, greater than 37.5 in 4.5%). No statistically significant differences among parents were noted for total score (mean ± SD 16.1 ± 12 in mothers and 18.3 ± 12.6 in fathers) or subscales, except the informed subscale (mean ± SD 16.7 ± 14.3 in mothers and 21.1 ± 16.6 in fathers). Parental self-report of prior knowledge about hypospadias and preference for neonatal circumcision correlated with lower Decisional Conflict Scale scores (p = 0.02 and p <0.01, respectively). No statistical association was found between score and parental education level (p = 0.7) or expertise of the counselor (staff vs pediatric urology fellow, p = 0.4). CONCLUSIONS These data describe the level of decisional conflict in couples agreeing to proceed with hypospadias repair, with no evidence of significant discrepancy between them. The novel description of factors related to decreased decisional conflict might help focus efforts aimed at minimizing difficulties encountered during the decision making process.

The impact of category, cytopathology and cytogenetics on development and progression of clonal and malignant myeloid transformation in inherited bone marrow failure syndromes

Inherited bone marrow failure syndromes are a group of rare, heterogeneous genetic disorders with a risk of clonal and malignant myeloid transformation including clonal marrow cytogenetic abnormalities, myelodysplastic syndrome and acute myeloid leukemia. The clinical characteristics, risk classification, prognostic factors and outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes are largely unknown. The aims of this study were to determine the impact of category, cytopathology and cytogenetics, the three components of the “Category Cytology Cytogenetics” classification of pediatric myelodysplastic syndrome, on the outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure. We used data from the Canadian Inherited Marrow Failure Registry. Among 327 patients with inherited bone marrow failure syndrome enrolled in the registry, the estimated risk of clonal and malignant myeloid transformation by the age of 18 years was 37%. The risk of clonal and malignant myeloid transformation varied according to the type of inherited bone marrow failure syndrome but was highest in Fanconi anemia. The development of clonal and malignant myeloid transformation significantly affected overall survival. Mortality varied based on cytopathological group. The largest group of patients had refractory cytopenia. Clonal marrow cytogenetic abnormalities were identified in 87% of patients with clonal and malignant myeloid transformation, and different cytogenetic groups had different impacts on disease progression. We conclude that category, cytopathology and cytogenetics in cases of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes have an important impact on outcome and that the classification of such cases should incorporate these factors.

Treatment of dyskeratosis congenita-associated pulmonary fibrosis with danazol

Individuals with Dyskeratosis Congenita (DC) are at increased risk for complications in variety of systems including pulmonary fibrosis. Idiopathic and DC‐associated pulmonary fibrosis are progressive and fatal disorders without known treatment. Here we describe, for the first time, marked improvement in the clinical and laboratory parameters of the pulmonary disease of a child who suffered from TINF2‐associated DC and severe pulmonary fibrosis after initiation of therapy with Danazol. We recommend that the clinical efficacy of Danazol in slowing down the progression of pulmonary fibrosis in patients with telomere‐related disorders is evaluated in prospective studies. Pediatr Pulmonol. 2015; 50:E48–E51.

Response to treatment with azacitidine in children with advanced myelodysplastic syndrome prior to hematopoietic stem cell transplantation.

Advanced myelodysplastic syndrome harbors a high risk of progression to acute myeloid leukemia and poor prognosis. In children, there is no established treatment to prevent or delay progression to leukemia prior to hematopoietic stem cell transplantation. Azacitidine is a hypomethylating agent, which was shown to slow progression to leukemia in adults with myelodysplastic syndrome. There is little data on the efficacy of azacitidine in children. We reviewed 22 pediatric patients with advanced myelodysplastic syndrome from a single center, diagnosed between January 2000 and December 2015. Of those, eight patients received off-label azacitidine before hematopoietic stem cell transplantation. A total of 31 cycles were administered and modification or delay occurred in four of them due to cytopenias, infection, nausea/vomiting, and transient renal impairment. Bone marrow blast percentages in azacitidine-treated patients decreased significantly from a median of 15% (range 9–31%) at the start of treatment to 5.5% (0–12%, P=0.02) before hematopoietic stem cell transplantation. Following azacitidine treatment, four patients (50%) achieved marrow remission, and none progressed. In contrast, three untreated patients (21.4%) had progressive disease characterized by >50% increase in blast counts or progression to leukemia. Azacitidine-treated patients had significantly increased 4-year event-free survival (P=0.04); predicted 4-year overall survival was 100% versus 69.3% in untreated patients (P=0.1). In summary, azacitidine treatment prior to hematopoietic stem cell transplantation was well tolerated in pediatric patients with advanced myelodysplastic syndrome, led to partial or complete bone marrow response in seven of eight patients (87.5%), and correlated with superior event-free survival in this cohort.

The Clinical Impact of Copy Number Variants in Inherited Bone Marrow Failure Syndromes

Inherited bone marrow failure syndromes comprise a genetically heterogeneous group of diseases with hematopoietic failure and a wide array of physical malformations. Copy number variants were reported in some inherited bone marrow failure syndromes. It is unclear what impact copy number variants play in patients evaluated for a suspected diagnosis of inherited bone marrow failure syndromes. Clinical and genetic data of 323 patients from the Canadian Inherited Marrow Failure Registry from 2001 to 2014, who had a documented genetic work-up, were analyzed. Cases with pathogenic copy number variants (at least 1 kilobasepairs) were compared to cases with other mutations. Genotype-phenotype correlations were performed to assess the impact of copy number variants. Pathogenic nucleotide-level mutations were found in 157 of 303 tested patients (51.8%). Genome-wide copy number variant analysis by single-nucleotide polymorphism arrays or comparative genomic hybridization arrays revealed pathogenic copy number variants in 11 of 67 patients tested (16.4%). In four of these patients, identification of copy number variant was crucial for establishing the correct diagnosis as their clinical presentation was ambiguous. Eight additional patients were identified to harbor pathogenic copy number variants by other methods. Of the 19 patients with pathogenic copy number variants, four had compound-heterozygosity of a copy number variant with a nucleotide-level mutation. Pathogenic copy number variants were associated with more extensive non-hematological organ system involvement (p = 0.0006), developmental delay (p = 0.006) and short stature (p = 0.04) compared to nucleotide-level mutations. In conclusion, a significant proportion of patients with inherited bone marrow failure syndromes harbor pathogenic copy number variants which were associated with a more extensive non-hematological phenotype in this cohort. Patients with a phenotype suggestive of inherited bone marrow failure syndromes but without identification of pathogenic nucleotide-level mutations should undergo specific testing for copy number variants.Blood disorders: impact of genomic structural variationCopy number variation in patients with inherited bone marrow failure syndromes (IBMFSs) is associated with more severe clinical symptoms. In addition to persistently low levels of red blood cells, white blood cells and/ or platelets, patients with IBMFSs also present varying degrees of physical malformations. Most cases are associated with single base-pair mutations in the DNA sequence, but Canadian researchers led by Yigal Dror at The Hospital for Sick Children in Toronto, have found that whole sections of the genome are deleted or repeated in an important proportion of patients. Those carrying copy number variants (CNV) presented more commonly with developmental delay, short stature and defects in more organ systems, than patients with point mutations. CNV analysis of patients with suspected IBMFSs could aid early disease evaluation and management.