Differential diagnosis of bone marrow failure syndromes guided by machine learning.

The choice to postpone treatment while awaiting genetic testing can result in significant delay in definitive therapies in patients with severe pancytopenia. Conversely, the misdiagnosis of inherited bone marrow failure (BMF) can expose patients to ineffectual and expensive therapies, toxic transplant conditioning regimens, and inappropriate use of an affected family member as a stem cell donor. To predict the likelihood of patients having acquired or inherited BMF, we developed a 2-step data-driven machine-learning model using 25 clinical and laboratory variables typically recorded at the initial clinical encounter. For model development, patients were labeled as having acquired or inherited BMF depending on their genomic data. Data sets were unbiasedly clustered, and an ensemble model was trained with cases from the largest cluster of a training cohort (n = 359) and validated with an independent cohort (n = 127). Cluster A, the largest group, was mostly immune or inherited aplastic anemia, whereas cluster B comprised underrepresented BMF phenotypes and was not included in the next step of data modeling because of a small sample size. The ensemble cluster A-specific model was accurate (89%) to predict BMF etiology, correctly predicting inherited and likely immune BMF in 79% and 92% of cases, respectively. Our model represents a practical guide for BMF diagnosis and highlights the importance of clinical and laboratory variables in the initial evaluation, particularly telomere length. Our tool can be potentially used by general hematologists and health care providers not specialized in BMF, and in under-resourced centers, to prioritize patients for genetic testing or for expeditious treatment.

Progression of liver disease and portal hypertension in dyskeratosis congenita and related telomere biology disorders.

BACKGROUND AND AIMS: Dyskeratosis congenita (DC) and related telomere biology disorders (TBD) are characterized by very short telomeres and multisystem organ involvement including liver disease. Our study aimed to characterize baseline hepatic abnormalities in patients with DC/TBD and determine risk factors associated with liver disease progression. APPROACH AND RESULTS: A retrospective review was performed on a cohort of 58 patients (39 males) with DC/TBD who were prospectively evaluated at a single institute from 2002 to 2019. The median age at initial assessment was 18 (1.4-67.6) years, and median follow-up duration was 6 (1.4-8.2) years. Patients with autosomal or X-linked recessive inheritance and those with heterozygous TINF2 DC were significantly younger, predominantly male, and more likely to have DC-associated mucocutaneous triad features and severe bone marrow failure compared with autosomal dominant-non- TINF2 DC/TBD patients. Liver abnormality (defined at baseline assessment by laboratory and/or radiological findings) was present in 72.4% of patients with predominantly cholestatic pattern of liver enzyme elevation. Clinically significant liver disease and portal hypertension developed in 17.2% of patients during the 6-year follow-up; this progression was mainly seen in patients with recessive or TINF2 -associated DC. Significant risk factors associated with progression included the presence of pulmonary or vascular disease. CONCLUSIONS: Our experience shows a high prevalence of cholestatic pattern of liver abnormality with progression to portal hypertension in patients with DC/TBD. Presence of pulmonary and/or vascular disease in patients with recessive or TINF2 DC was an important predictor of liver disease progression, suggesting the need for increased vigilance and monitoring for complications in these patients.

Disease progression and clinical outcomes in telomere biology disorders.

Dyskeratosis congenita related telomere biology disorders (DC/TBDs) are characterized by very short telomeres caused by germline pathogenic variants in telomere biology genes. Clinical presentations can affect all organs, and inheritance patterns include autosomal dominant (AD), autosomal recessive (AR), X-linked (XLR), or de novo. This study examined the associations between mode of inheritance with phenotypes and long-term clinical outcomes. Two hundred thirty-one individuals with DC/TBDs (144 male, 86.6% known genotype, median age at diagnosis 19.4 years [range 0 to 71.6]), enrolled in the National Cancer Institute's Inherited Bone Marrow Failure Syndrome Study, underwent detailed clinical assessments and longitudinal follow-up (median follow-up 5.2 years [range 0 to 36.7]). Patients were grouped by inheritance pattern, considering AD-nonTINF2, AR/XLR, and TINF2 variants separately. Severe bone marrow failure (BMF), severe liver disease, and gastrointestinal telangiectasias were more prevalent in AR/XLR or TINF2 disease, whereas pulmonary fibrosis developed predominantly in adults with AD disease. After adjusting for age at DC/TBD diagnosis, we observed the highest cancer risk in AR/XLR individuals. At last follow-up, 42% of patients were deceased with a median overall survival (OS) of 52.8 years (95% confidence interval [CI] 45.5-57.6), and the hematopoietic cell or solid organ transplant-free median survival was 45.3 years (95% CI 37.4-52.1). Significantly better OS was present in AD vs AR/XLR/TINF2 disease (P < .01), while patients with AR/XLR and TINF2 disease had similar survival probabilities. This long-term study of the clinical manifestations of DC/TBDs creates a foundation for incorporating the mode of inheritance into evidence-based clinical care guidelines and risk stratification in patients with DC/TBDs. This trial was registered at www.clinicaltrials.gov as #NCT00027274.

The distribution and accumulation of the shortest telomeres in telomere biology disorders.

Individuals with telomere biology disorders (TBDs) have very short telomeres, high risk of bone marrow failure (BMF), and reduced survival. Using data from TBD patients, a mean leukocyte Southern blot telomere length (TL) of 5 kilobases (kb) was estimated as the 'telomere brink' at which human survival is markedly reduced. However, the shortest telomere, not the mean TL, signals replicative senescence. We used the Telomere Shortest Length Assay (TeSLA) to tally TL of all 46 chromosomes in blood-derived DNA and examined its relationship with TBDs. Patients (n = 18) had much shorter mean TL (TeSmTL) (2.54 ± 0.41 kb vs. 4.48 ± 0.52 kb, p < 0.0001) and more telomeres <3 kb than controls (n = 22) (70.43 ± 8.76% vs. 33.05 ± 6.93%, p < 0.0001). The proportion of ultrashort telomeres (<1.6 kb) was also higher in patients than controls (39.29 ± 10.69% vs. 10.40 ± 4.09%, p < 0.0001). TeS <1.6 kb was associated with severe (n = 11) compared with non-severe (n = 7) BMF (p = 0.027). Patients with multi-organ manifestations (n = 10) had more telomeres <1.6 kb than those with one affected organ system (n = 8) (p = 0.029). Findings suggest that TBD clinical manifestations are associated with a disproportionately higher number of haematopoietic cell telomeres reaching a telomere brink, whose length at the single telomere level is yet to be determined.

Next-generation sequencing errors due to genetic variation in WRAP53 encoding TCAB1 on chromosome 17.

Next-generation sequencing (NGS) is a valuable tool, but has limitations in sequencing through repetitive runs of single nucleotides (homopolymers). Pathogenic germline variants in WRAP53 encoding telomere Cajal body protein 1 (TCAB1) are a known cause of dyskeratosis congenita. We identified a significant NGS error in WRAP53, c.1562dup, p.Ala522Glyfs*8 (rs755116516 G>-/GG/GGG) that did not validate by Sanger sequencing. This error occurs because rs755116516 G>-/GG/GGG (Chr17:7,606,714) is polymorphic, and variants at this site challenge the ability of NGS to accurately call the correct number of nucleotides in a homopolymer run. This was further complicated by the fact that chr17:7,606,721 (rs769202794) is multiallelic G>A, C, T, and that chr17:7,606,722 is also multiallelic (rs7640C>A/G/T and rs373064567C>delC). In addition to the expert interpretation of potentially clinically actionable variants, it recommended that all variants in regions of the genome with homopolymers be validated by Sanger sequencing before clinical action.

Constitutional SAMD9L mutations cause familial myelodysplastic syndrome and transient monosomy 7.

Familial myelodysplastic syndromes arise from haploinsufficiency of genes involved in hematopoiesis and are primarily associated with early-onset disease. Here we describe a familial syndrome in seven patients from four unrelated pedigrees presenting with myelodysplastic syndrome and loss of chromosome 7/7q. Their median age at diagnosis was 2.1 years (range, 1-42). All patients presented with thrombocytopenia with or without additional cytopenias and a hypocellular marrow without an increase of blasts. Genomic studies identified constitutional mutations (p.H880Q, p.R986H, p.R986C and p.V1512M) in the SAMD9L gene on 7q21, with decreased allele frequency in hematopoiesis. The non-random loss of mutated SAMD9L alleles was attained via monosomy 7, deletion 7q, UPD7q, or acquired truncating SAMD9L variants p.R1188X and p.S1317RfsX21. Incomplete penetrance was noted in 30% (3/10) of mutation carriers. Long-term observation revealed divergent outcomes with either progression to leukemia and/or accumulation of driver mutations (n=2), persistent monosomy 7 (n=4), and transient monosomy 7 followed by spontaneous recovery with SAMD9L-wildtype UPD7q (n=2). Dysmorphic features or neurological symptoms were absent in our patients, pointing to the notion that myelodysplasia with monosomy 7 can be a sole manifestation of SAMD9L disease. Collectively, our results define a new subtype of familial myelodysplastic syndrome and provide an explanation for the phenomenon of transient monosomy 7. Registered at: www.clinicaltrials.gov; #NCT00047268.

Germline biallelic BRCA2 pathogenic variants and medulloblastoma: an international cohort study.

Constitutional heterozygous pathogenic variants in genes coding for some components of the Fanconi anemia-BRCA signaling pathway, which repairs DNA interstrand crosslinks, represent risk factors for common cancers, including breast, ovarian, pancreatic and prostate cancer. A high cancer risk is also a main clinical feature in patients with Fanconi anemia (FA), a rare condition characterized by bone marrow failure, endocrine and physical abnormalities. The mainly recessive condition is caused by germline pathogenic variants in one of 21 FA-BRCA pathway genes. Among patients with FA, the highest cancer risks are observed in patients with biallelic pathogenic variants in BRCA2 or PALB2. These patients develop a range of embryonal tumors and leukemia during the first decade of life, however, little is known about specific clinical, genetic and pathologic features or toxicities. Here, we present genetic, clinical, pathological and treatment characteristics observed in an international cohort of eight patients with FA due to biallelic BRCA2 pathogenic variants and medulloblastoma (MB), an embryonal tumor of the cerebellum. Median age at MB diagnosis was 32.5 months (range 7-58 months). All patients with available data had sonic hedgehog-MB. Six patients received chemotherapy and one patient also received proton radiation treatment. No life-threatening toxicities were documented. Prognosis was poor and all patients died shortly after MB diagnosis (median survival time 4.5 months, range 0-21 months) due to MB or other neoplasms. In conclusion, MB in patients with biallelic BRCA2 pathogenic variants is a lethal disease. Future experimental treatments are necessary to help these patients.

Liver disease and transplantation in telomere biology disorders: An international multicenter cohort.

BACKGROUND: Patients with telomere biology disorders (TBD) develop hepatic disease, including hepatitis, cirrhosis, and hepatopulmonary syndrome. No specific treatment exists for TBD-related liver disease, and the role of liver transplantation (LT) remains controversial. Our study objectives were to describe the clinical characteristics, management, and outcomes in patients with TBD-related liver disease, and their LT outcomes. METHODS: Data from 83 patients with TBD-associated liver disease were obtained from 17 participating centers in the Clinical Care Consortium of Telomere-Associated Ailments and by self-report for our retrospective, multicenter, international cohort study. RESULTS: Group A ("Advanced") included 40 patients with advanced liver disease. Of these, 20 underwent LT (Group AT). Group M ("Mild") included 43 patients not warranting LT evaluation, none of whom were felt to be medically unfit for liver transplantation. Supplemental oxygen requirement, pulmonary arteriovenous malformation, hepatopulmonary syndrome, and higher bilirubin and international normalized ratio values were associated with Group A. Other demographics, clinical manifestations, and laboratory findings were similar between groups. Six group A patients were declined for LT; 3 died on the waitlist. Median follow-up post-LT was 2.9 years (range 0.6-13.2 y). One-year survival post-LT was 73%. Median survival post-LT has not been reached. Group AT patients had improved survival by age compared to all nontransplant patients (log-rank test p = 0.02). Of 14 patients with pretransplant hypoxemia, 8 (57%) had improved oxygenation after transplant. CONCLUSIONS: LT recipients with TBD do not exhibit excessive posttransplant mortality, and LT improved respiratory status in 57%. A TBD diagnosis should not exclude LT consideration.

Clinical manifestations of telomere biology disorders in adults.

Telomere biology disorders (TBDs) are a spectrum of inherited bone marrow failure syndromes caused by impaired telomere function due to pathogenic germline variants in genes involved in telomere maintenance. TBDs can affect many organ systems and are often thought of as diseases of childhood. However, TBDs may present in mid- or even late adulthood with features similar to but not always the same as the childhood-onset TBDs. Adult-onset TBDs are often cryptic with isolated pulmonary, liver, or hematologic disease, or cancer, and may lack the classic disease-defining triad of abnormal skin pigmentation, nail dysplasia, and oral leukoplakia. Diagnostics include detection of very short leukocyte telomeres and germline genetic testing. Notably, adult-onset TBDs may show telomeres in the 1st to 10th percentile for age, and some cases may not have an identifiable genetic cause. TBD genetic etiology includes all modes of inheritance, with autosomal dominant the most frequent in adult-onset disease. Variable symptom onset due to incomplete penetrance, variable expressivity, and genetic anticipation add to the diagnostic challenges. Adult-onset TBDs are likely underrecognized, but their correct identification is of utmost importance, since affected patients are faced with numerous clinical complications, including but not limited to an increased risk of malignancies requiring close surveillance for early detection. Currently lung, liver, or hematopoietic cell transplants are the only curative therapeutic approaches but can be complicated by comorbidities, despite improved medical care. This review highlights the challenges of identifying adult-onset TBDs and addresses currently recommended clinical screening measures and therapy options.

Research on Rare Diseases in Germany - The cancer predisposition syndrome registry.

Background: Cancer predisposition syndromes (CPS) are rare diseases that are associated with an increased risk of cancer due to genetic alterations. At least 8 % of all cases of childhood cancer are attributable to CPS [1, 2]. The CPS registry was launched in 2017 to learn more about CPS and to improve the care to those afflicted by these diseases. Methods: This is an internationally networked registry with associated accompanying studies that investigate cancer risks and spectra, the possibilities of cancer prevention, early detection and therapy. Results: For several of these syndromes, new insights into the cancer risks and cancer types as well as factors modifying cancer risk have been gained. In addition, experimental, psycho-oncological, preclinical and clinical studies were initiated. Conclusions: The CPS registry is an example of how progress can be made within a short period of time to the benefit of individuals with rare diseases through systematic data collection and research.

Influence of dichloroacetate (DCA) on lactate production and oxygen consumption in neuroblastoma cells: is DCA a suitable drug for neuroblastoma therapy?

Many cancer cells metabolize glucose preferentially via pyruvate to lactate instead to CO(2) and H(2)O (oxidative phosphorylation) even in the presence of oxygen (Warburg effect). Dichloroacetate (DCA) is a drug which is able to shift pyruvate metabolism from lactate to acetyl-CoA (tricarboxylic acid cycle) by indirect activation of pyruvate dehydrogenase (PDH). This can subsequently lead to an increased flow of oxygen in the respiratory chain, associated with enhanced generation of reactive oxygen species (ROS) which may cause apoptosis. In order to investigate if DCA may be suitable for neuroblastoma therapy, it was investigated on three human neuroblastoma cell lines whether DCA can reduce lactate production and enhance oxygen consumption. The data show, that DCA (in the low millimolar range) is able to reduce lactate production, but there was only a slight shift to increased oxygen consumption and almost no effect on cell vitality, proliferation and apoptosis of the three cell lines investigated. Therefore, DCA at low millimolar concentrations seems to be only of minor efficacy for neuroblastoma treatment.

Clinical evolution, genetic landscape and trajectories of clonal hematopoiesis in SAMD9/SAMD9L syndromes.

Germline SAMD9 and SAMD9L mutations (SAMD9/9Lmut) predispose to myelodysplastic syndromes (MDS) with propensity for somatic rescue. In this study, we investigated a clinically annotated pediatric MDS cohort (n = 669) to define the prevalence, genetic landscape, phenotype, therapy outcome and clonal architecture of SAMD9/9L syndromes. In consecutively diagnosed MDS, germline SAMD9/9Lmut accounted for 8% and were mutually exclusive with GATA2 mutations present in 7% of the cohort. Among SAMD9/9Lmut cases, refractory cytopenia was the most prevalent MDS subtype (90%); acquired monosomy 7 was present in 38%; constitutional abnormalities were noted in 57%; and immune dysfunction was present in 28%. The clinical outcome was independent of germline mutations. In total, 67 patients had 58 distinct germline SAMD9/9Lmut clustering to protein middle regions. Despite inconclusive in silico prediction, 94% of SAMD9/9Lmut suppressed HEK293 cell growth, and mutations expressed in CD34+ cells induced overt cell death. Furthermore, we found that 61% of SAMD9/9Lmut patients underwent somatic genetic rescue (SGR) resulting in clonal hematopoiesis, of which 95% was maladaptive (monosomy 7 ± cancer mutations), and 51% had adaptive nature (revertant UPD7q, somatic SAMD9/9Lmut). Finally, bone marrow single-cell DNA sequencing revealed multiple competing SGR events in individual patients. Our findings demonstrate that SGR is common in SAMD9/9Lmut MDS and exemplify the exceptional plasticity of hematopoiesis in children.

H(2)O(2)-mediated cytotoxicity of pharmacologic ascorbate concentrations to neuroblastoma cells: potential role of lactate and ferritin.

By intravenous (but not oral) application of ascorbate, millimolar serum concentrations can be reached, which are preferentially cytotoxic to cancer cells. Cytotoxicity is mediated by transition metal-dependent generation of H(2)O(2) in the interstitial space. In this study, the sensitivity of neuroblastoma cells (Kelly, SK-N-SH) to ascorbate and H(2)O(2) and their defense mechanisms against H(2)O(2) were investigated. Since aerobic glycolysis (the Warburg effect) is a feature of many tumour cells, their glucose consumption and lactate production were monitored. Furthermore, synthesis and release of ferritin by neuroblastoma cells were analysed in order to examine whether ferritin is possibly an iron source for H(2)O(2) generation. Ascorbate (0.6-5.0 mM) and H(2)O(2) (25-100 muM) were found to be similarly cytotoxic to Kelly and SK-N-SH cells. In each case, cytotoxicity increased if cell concentrations decreased, in accordance with low cell concentrations having lower capacities to detoxify H(2)O(2). Kelly and SK-N-SH cells produced and released remarkable amounts of lactate and ferritin. We propose the selective cytotoxicity of high dose ascorbate to tumour cells to be due to the preferential generation of H(2)O(2) in the acidic and ferritin-rich tumour microenvironment, combined with reduced defense systems against H(2)O(2) as a consequence of aerobic glycolysis.

An update on the biology and management of dyskeratosis congenita and related telomere biology disorders.

Introduction: Telomere biology disorders (TBDs) encompass a group of illnesses caused by germline mutations in genes regulating telomere maintenance, resulting in very short telomeres. Possible TBD manifestations range from complex multisystem disorders with onset in childhood such as dyskeratosis congenita (DC), Hoyeraal-Hreidarsson syndrome, Revesz syndrome and Coats plus to adults presenting with one or two DC-related features.Areas covered: The discovery of multiple genetic causes and inheritance patterns has led to the recognition of a spectrum of clinical features affecting multiple organ systems. Patients with DC and associated TBDs are at high risk of bone marrow failure, cancer, liver and pulmonary disease. Recently, vascular diseases, including pulmonary arteriovenous malformations and gastrointestinal telangiectasias, have been recognized as additional manifestations. Diagnostics include detection of very short leukocyte telomeres and germline genetic testing. Hematopoietic cell transplantation and lung transplantation are the only current therapeutic modalities but are complicated by numerous comorbidities. This review summarizes the pathophysiology underlying TBDs, associated clinical features, management recommendations and therapeutic options.Expert opinion: Understanding TBDs as complex, multisystem disorders with a heterogenous genetic background and diverse phenotypes, highlights the importance of clinical surveillance and the urgent need to develop new therapeutic strategies to improve health outcomes.

Genotype and phenotype spectrum of NRAS germline variants.

RASopathies comprise a group of disorders clinically characterized by short stature, heart defects, facial dysmorphism, and varying degrees of intellectual disability and cancer predisposition. They are caused by germline variants in genes encoding key components or modulators of the highly conserved RAS-MAPK signalling pathway that lead to dysregulation of cell signal transmission. Germline changes in the genes encoding members of the RAS subfamily of GTPases are rare and associated with variable phenotypes of the RASopathy spectrum, ranging from Costello syndrome (HRAS variants) to Noonan and Cardiofaciocutaneous syndromes (KRAS variants). A small number of RASopathy cases with disease-causing germline NRAS alterations have been reported. Affected individuals exhibited features fitting Noonan syndrome, and the observed germline variants differed from the typical oncogenic NRAS changes occurring as somatic events in tumours. Here we describe 19 new cases with RASopathy due to disease-causing variants in NRAS. Importantly, four of them harbored missense changes affecting Gly12, which was previously described to occur exclusively in cancer. The phenotype in our cohort was variable but well within the RASopathy spectrum. Further, one of the patients (c.35G>A; p.(Gly12Asp)) had a myeloproliferative disorder, and one subject (c.34G>C; p.(Gly12Arg)) exhibited an uncharacterized brain tumour. With this report, we expand the genotype and phenotype spectrum of RASopathy-associated germline NRAS variants and provide evidence that NRAS variants do not spare the cancer-associated mutation hotspots.