PersonALL: a genetic scoring guide for personalized risk assessment in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. METHODS: Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. RESULTS: CNAs were detected in 93.8% (n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status (IKZF1normal, IKZF1del and IKZF1plus) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low (n = 215): 86.3%, IKAROS-medium (n = 27): 57.4% and IKAROS-high (n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients (n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients (n = 103) showed high (n = 74) or ultra-poor (n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). CONCLUSIONS: PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

An Ultra-Rare Manifestation of an X-Linked Recessive Disorder: Duchenne Muscular Dystrophy in a Female Patient.

Duchenne muscular dystrophy (DMD) is the most common inherited muscle dystrophy. Patients are characterized by muscle weakness, gross motor delay, and elevated serum creatinine kinase (CK) levels. The disease is caused by mutations in the DMD gene located on the X chromosome. Due to the X-linked recessive inheritance pattern, DMD most commonly affects males, who are generally diagnosed between the age of 3-5 years. Here we present an ultra-rare manifestation of DMD in a female patient. Cytogenetic examination showed that she has a t(X;10)(p21.1;p12.1) translocation, which turned out to affect the DMD gene with one of the breakpoints located in exon 54 (detected by genome sequencing). The X-inactivation test revealed skewed X-inactivation (ratio 99:1). Muscle histology and dystrophin immunohistochemistry showed severe dystrophic changes and highly reduced dystrophin expression, respectively. These results, in accordance with the clinical picture and a highly elevated serum CK, led to the diagnosis of DMD. In conclusion, although in very rare cases, DMD can manifest in female patients as well. In this case, a balanced X-autosome reciprocal translocation disrupts the DMD gene and skewed X-inactivation leads to the manifestation of the DMD phenotype.

Hereditary and non-hereditary etiologies associated with extensive brain calcification: case series.

Cerebral calcification may be caused by several potentially treatable conditions, however, in most cases it does not receive special attention in clinical practice. From the point of view of etiology, the diseases associated with cerebral calcification can be divided into two main groups: idiopathic (mostly incurable) and secondary (potentially treatable). The first group includes mainly the hereditary diseases identified before 2021 (primary familial brain calcification subtypes, previously known as Fahr's disease or Fahr's syndrome). In contrast, the second group includes diseases with cerebral calcification that develop generally as a consequence of metabolic/endocrine/autoimmune abnormalities. The aim of our research was to present hereditary and non-hereditary etiologies associated with extensive brain calcification. We compare the detailed clinical, radiological and laboratory results of 6 patients with prominent cerebral calcification identified in our clinic in the last 3 years (idiopathic and secondary etiologies as well). Our research draws attention to the complexity of the etiologies in the context of cerebral calcification. We recommend, beside NGS-based sequence analyses, the application of array comparative genomic hybridization as well, to identify potential genetic etiologies associated with brain calcification.

A novel flow cytometric method for enhancing acute promyelocytic leukemia screening by multidimensional dot-plots.

Acute promyelocytic leukemia (APL) is generally characterized by t(15;17)(q24;q21). In some cases, the classic translocation cannot be identified by conventional methods, since the PML-RARA fusion protein results from complex, variant, or cryptic translocation. The diagnostic algorithm of APL starts with screening methods, such as flow cytometry (FC), followed by fluorescence in situ hybridization or polymerase chain reaction to confirm the diagnosis. Our aim was to develop a novel protocol for analyzing APL samples based on multidimensional dot-plots that can provide comprehensive information about several markers at the same time. The protocol included four optimized multidimensional dot-plots, which were tested by retrospective reanalysis of FC results in APL (n = 8) and non-APL (n = 12) acute myeloid leukemia (AML) cases. After predicting the potential position of hypergranular- and microgranular-type aberrant promyelocytes, the percentages of blast populations were examined within the gates in all AML cases. The percentage of blasts in each predefined gate was well above the cut-off value (95%) in APL cases in all tubes. In non-APL AML cases, the percentage of blasts in the same gates never reached the cut-off value in all investigated tubes, and even when it did in a single tube, the pattern was markedly different from that observed in APL cases. In conclusion, multidimensional dot-plots can be used for screening APL even in cryptic APL cases, although reproducibility across several laboratories would require standardization of antibodies and fluorochromes. This easy-to-use and quick method can support the diagnosis of APL and the prompt initiation of the appropriate treatment.

The importance of the multiplex ligation-dependent probe amplification in the identification of a novel two-exon deletion of the NR5A1 gene in a patient with 46,XY differences of sex development.

Gonadal dysgenesis (GD) is a rare cause of differences of sex development (DSD) with highly variable clinical and genetic conditions. Although identification of the causative genetic alterations can offer a clearer prognosis and personalized management to patients, more than 50% of the DSD cases still do not have an accurate genetic diagnosis. NR5A1 (previously known as SF-1), is a transcriptional regulator of genes required for normal development and functional maintenance of the gonads and the adrenal glands. Nucleotide sequence variants of the NR5A1 gene have been reported in numerous patients with GD with or without adrenal failure, however, microdeletion or partial deletion in the NR5A1 gene have been described only in a few GD cases. In this case study, we present a subject with female phenotype, mild clitoromegaly, partial GD and normal adrenal function. Cytogenetic analysis revealed a 46,XY SRY + karyotype. Microarray analysis did not identify pathogenic copy number variations, nor did panel sequencing of the most common DSD genes. Subsequently, multiplex ligation-dependent probe amplification (MLPA) was performed to test for small deletion/duplication of the most frequently affected genes associated with GD. Using this method, we have identified a novel heterozygous deletion involving exons 5 and 6 of the NR5A1 gene as the cause of abnormal sexual development of the patient. This report expands our knowledge about the range and pathogenetic role of NR5A1 mutations associated with partial gonadal dysgenesis in 46,XY DSD. Furthermore, our data emphasises the indispensable role of MLPA in the diagnosis of DSD with unclear etiology.

Clinical and genetic characteristics of craniosynostosis in Hungary.

Craniosynostosis, the premature closure of cranial sutures, is a common craniofacial disorder with heterogeneous etiology and appearance. The purpose of this study was to investigate the clinical and molecular characteristics of craniosynostoses in Hungary, including the classification of patients and the genetic analysis of the syndromic forms. Between 2006 and 2012, 200 patients with craniosynostosis were studied. Classification was based on the suture(s) involved and the associated clinical features. In syndromic cases, genetic analyses, including mutational screening of the hotspot regions of the FGFR1, FGFR2, FGFR3, and TWIST1 genes, karyotyping and FISH study of TWIST1, were performed. The majority (88%) of all patients with craniosynostosis were nonsyndromic. The sagittal suture was most commonly involved, followed by the coronal, metopic, and lambdoid sutures. Male, twin gestation, and very low birth weight were risk factors for craniosynostosis. Syndromic craniosynostosis was detected in 24 patients. In 17 of these patients, Apert, Crouzon, Pfeiffer, Muenke, or Saethre-Chotzen syndromes were identified. In one patient, multiple-suture craniosynostosis was associated with achondroplasia. Clinical signs were not typical for any particular syndrome in six patients. Genetic abnormalities were detected in 18 syndromic patients and in 8 relatives. In addition to 10 different, known mutations in FGFR1,FGFR2 or FGFR3, one novel missense mutation, c.528C>G(p.Ser176Arg), was detected in the TWIST1 gene of a patient with Saethre-Chotzen syndrome. Our results indicate that detailed clinical assessment is of paramount importance in the classification of patients and allows indication of targeted molecular testing with the highest possible diagnostic yield.

[Results of clinical and genetic diagnosis of rare diseases in the Eastern region of Hungary (2007-2013)]. / Ritka genetikai betegségek klinikai és genetikai diagnosztikájában szerzett tapasztalataink a kelet-magyarországi régióban (2007-2013).

INTRODUCTION: 80% of rare diseases have a genetic origin, and 50% manifest themselves as congenital anomalies. Their adequate health care includes early recognition of genetic anomalies and prevention of recurrence. AIM: The aims of the authors were to provide correct diagnoses to patients with multiple congenital anomalies with or without mental retardation attending to the outpatient clinic of the Clinical Genetics Center at the University of Debrecen in the time interval between August 1, 2007 and March 31, 2013, establish the possibility of prenatal diagnosis, assess the distribution of different genetic mechanisms in the background of rare genetic diseases, compare them with international data, and develop an algorithm for the diagnostic approach of rare genetic diseases applicable in Hungary. METHOD: Clinical data and genetic results of patients were evaluated, and patients were categorized into one of the ten proposed etiological groups, based on which the distribution of genetic causes was defined. RESULTS: Clinical diagnosis was achieved in 64.3% of patients, confirmed genetic diagnosis in 37.8%, while 35.7% of patients remained undiagnosed. Several dysmorphic syndromes and metabolic disorders were first diagnosed in Hungary, two of which unique in the literature. CONCLUSIONS: In the centre of the authors the diagnostic effectiveness of chromosome aberrations exceeds the international standards, that of known microdeletions and dysmorphic syndromes meets international data, and the genetic diagnosis of mendelian disorders and submicroscopic copy number changes remain below international figures.

[Deletion 15q26 syndrome]. / 15q26-microdeletio-szindróma.

The association of short stature, microcephaly, congenital cardiac anomaly and intellectual deficit should always raise the suspicion of chromosomal etiology. If G-banded karyotyping fails to detect large chromosomal aberrations, array comparative genomic hybridization (array CGH) should be performed to screen for submicroscopic pathological copy number changes. The authors present a six-year-old girl whose symptoms arose from a 4.1 Mb loss in the 15q26.2-26.3 telomeric region. The syndrome is characterized by a resistance to the insulin-like growth factor 1 - in our case the increased level of the insulin-like growth factor 1 together with the persistent longitudinal growth failure was an important finding and differential diagnostic feature. A brief overview of the literature is provided.

[Treatment of acute myeloid leukemia -- a single center experience (2007-2013)]. / Heveny myeloid leukaemiás betegeink kezelésével szerzett tapasztalataink (2007-2013).

INTRODUCTION: Mortality of acute myeloid leukemia is still 60-70% in young (<60 years) adults and 90% in elderly (≥60 years) patients. AIM: The aim of the authors was to analyse the outcome of treatment in their patients with acute myeloid leukemia. METHOD: From 2007 to 2013, 173 patients with acute myeloid leukemia were treated. Patients were classified according to the European LeukemiaNet prognostic guideline. Association between mortality and the type of acute myeloid leukemia (secondary or primary), dose of daunoblastin at induction of treatment, and the rate of minimal residual disease were investigated. RESULTS: The 5-year survival probability was 25% in young adults and 2% in the elderly. The survival was significantly influenced by these prognostic factors. The 5-year survival rate was 50% in the young, favorable prognostic group. The 90 mg/m2 daunoblastin dose was found to be beneficial. Addition of bortezomib to the standard induction protocol had an additional beneficial effect. CONCLUSIONS: The speed and depth of the response to induction therapy, and the initial white blood cell count had an apparent effect on survival.

Enhancing HLA-B27 antigen detection: Leveraging machine learning algorithms for flow cytometric analysis.

As the association of human leukocyte antigen B27 (HLA-B27) with spondylarthropathies is widely known, HLA-B27 antigen expression is frequently identified using flow cytometric or other techniques. Because of the possibility of cross-reaction with off target antigens, such as HLA-B7, each flow cytometric technique applies a "gray zone" reserved for equivocal findings. Our aim was to use machine learning (ML) methods to classify such equivocal data as positive or negative. Equivocal samples (n = 99) were selected from samples submitted to our institution for clinical evaluation by HLA-B27 antigen testing. Samples were analyzed by flow cytometry and polymerase chain reaction. Features of histograms generated by flow cytometry were used to train and validate ML methods for classification as logistic regression (LR), decision tree (DT), random forest (RF) and light gradient boost method (GBM). All evaluated ML algorithms performed well, with high accuracy, sensitivity, specificity, as well as negative and positive predictive values. Although, gradient boost approaches are proposed as high performance methods; nevertheless, their effectiveness may be lower for smaller sample sizes. On our relatively smaller sample set, the random forest algorithm performed best (AUC: 0.92), but there was no statistically significant difference between the ML algorithms used. AUC values for light GBM, DT, and LR were 0.88, 0.89, 0.89, respectively. Implementing these algorithms into the process of HLA-B27 testing can reduce the number of uncertain, false negative or false positive cases, especially in laboratories where no genetic testing is available.

[Citogenetic and molecular genetic studies in infertility in eastern Hungary]. / Infertilitásban végzett citogenetikai és molekuláris genetikai vizsgálatok a kelet-magyarországi régióban.

INTRODUCTION: In developed countries 10-15% of the couples are affected by infertility. In half of them genetic factors can be identified. AIMS: We studied genetic alterations in infertility in Hungarian patients. METHODS: Cyogenetic analyses were performed in 195 females and 305 males. In 17 females FMR1 mutations, in 150 males Y microdeletions, and aneuploidy were studied in the sperm of 28 males. In a carrier male sperm meiotic segregation was studied. RESULTS: The most common aberrations in females were X chromosome aneuploidia and inversion (3.6%), while the same in males Klinefelter-syndrome (3.3%) and autosomal translocations (2%). In two females FMR1 premutation was found. While Y microdeletions were identified only in azoospermic and severe oligozoospermic men, partial microdeletions could also be detected in normozoospermic males. A higher aberration rate was found in cases with abnormality in both the number and motility of sperm. In a male patient with 46,XY,t(3;6)(q21;q23) karyotype, 53.2% of spem carried unbalanced chromosome assortment. CONCLUSIONS: Knowledge of abnormalities may help in genetic counseling and choosing the most effective reproduction technique.

Next-Generation Sequencing-Based Genomic Profiling of Children with Acute Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality, with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML Berlin-Frankfurt-Münster protocols. Targeted next-generation sequencing of 54 genes revealed 17 genes that were recurrently mutated in >5% of patients. Considerable differences were observed in the mutational profiles compared with previous studies, as BCORL1, CUX1, KDM6A, PHF6, and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in the BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, and WT1) mutations were found to be associated with induction failure and shorter event-free survival, suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% versus 44.4%) and transcription factors (35.1% versus 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identify previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.

Subtelomeric 6.7 Mb trisomy 10p and 5.6 Mb monosomy 21q detected by FISH and array-CGH in three related patients.

Cryptic subtelomeric chromosomal aberrations are responsible for 5-10% of moderate/severe and 1% of mild intellectual disability. Unbalanced subtelomeric chromosomal rearrangements result in variable phenotypes which seem to be highly influenced by both the size of the duplication/deletion and the chromosomes involved in the translocation. We report on three related patients with moderate intellectual disability, language delay, hypotonia, facial dysmorphism, cardiac anomalies, scoliosis, and kyphosis in whom a familial (maternal) unbalanced submicroscopic translocation was found by subtelomeric fluorescence in situ hybridization (FISH). This rearrangement resulted in a partial trisomy 10pter and partial monosomy 21qter. The karyotype was 46,XY.ish der(21)t(10;21)(p14;q22.2). Confirmation of a 6.7 Mb size distal duplication of the p15.3-14 region of chromosome 10 and a 5.6 Mb distal deletion of the q22.2-22.3 region of chromosome 21 was obtained by array-CGH. To our best knowledge, such a composition of subtelomeric unbalanced translocations has not yet been published. Detection of this aberration in successive pregnancies of carrier members of the family by prenatal FISH could prevent the recurrence of the disease. Furthermore, detection of the rearrangements and identification of genes located in the chromosomal regions involved might be of interest.

Correlation study between sperm concentration, hyaluronic acid-binding capacity and sperm aneuploidy in Hungarian patients.

Infertile men with low sperm concentration and/or less motile spermatozoa have an increased risk of producing aneuploid spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding may reduce genetic risks such as chromosomal rearrangements and numerical aberrations. Fluorescence in-situ hybridization (FISH) has been used to evaluate the presence of aneuploidies. This study examined spermatozoa of 10 oligozoospermic, 9 asthenozoospermic, 9 oligoasthenozoospermic and 17 normozoospermic men by HA binding and FISH. Mean percentage of HA-bound spermatozoa in the normozoospermic group was 81%, which was significantly higher than in the oligozoospermic (P<0.001), asthenozoospermic (P<0.001) and oligoasthenozoospermic (P<0.001) groups. Disomy of sex chromosomes (P=0.014) and chromosome 17 (P=0.0019), diploidy (P=0.03) and estimated numerical chromosome aberrations (P=0.004) were significantly higher in the oligoasthenozoospermic group compared with the other groups. There were statistically significant relationships (P<0.001) between sperm concentration and HA binding (r=0.658), between sperm concentration and estimated numerical chromosome aberrations (r=-0.668) and between HA binding and estimated numerical chromosome aberrations (r=-0.682). HA binding and aneuploidy studies of spermatozoa in individual cases allow prediction of reproductive prognosis and provision of appropriate genetic counselling. Infertile men with normal karyotypes and low sperm concentrations and/or less motile spermatozoa have significantly increased risks of producing aneuploid (diminished mature) spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding, based on a binding between sperm receptors for zona pellucida and HA, may reduce the potential genetic risks such as chromosomal rearrangements and numerical aberrations. In the present study we examined sperm samples of 45 men with different sperm parameters by HA-binding assay and fluorescence in-situ hybridization (FISH). Mean percentage of HA-bound spermatozoa in the normozoospermic group was significantly higher than the oligozoospermic, the asthenozoospermic and the oligoasthenozoospermic groups. Using FISH, disomy of sex chromosomes and chromosome 17, diploidy and estimated numerical chromosome aberration frequencies were significantly higher in the oligoasthenozoospermic group compared with the three other groups. A significant positive correlation was found between the sperm concentration and the HA-binding capacity, and significant negative correlations between the sperm concentration and the estimated numerical chromosomes aberrations as well as between the HA-binding ability and the estimated numerical chromosome aberrations were identified. We conclude that HA-binding assay and sperm aneuploidy study using FISH may help to predict the reproductive ability of selected infertile male patients and to provide appropriate genetic counselling.

[Dysgenetic male pseudohermaphroditism]. / Dysgeneticus pseudohermaphroditismus masculinus.

The authors report a case of a dysgenetic male pseudohermaphroditism with a 45,X/46,XY karyotype in a mosaic form, which was diagnosed in an infant. The one-week-old infant was evaluated because of proximal hypospadias and retention of the right testis. The results of hormonal tests were the followings: serum FSH 5.2 mU/ml; LH: 2.0 mU/ml; testosterone: 144.3 ng/dl; androstendione: 0.42 µg/l; 17-hydroxyprogesterone: 1.12 ng/ml. Chromosomal analysis revealed 45,X/46,XY karyotype. Fluorescent in vitro hybridization showed that 51% of the lymphocytes had the Y chromosome and the SRY gene. Analysis of the SRY showed no deletion in the AZF a,b,c regions. Pelvic magnetic resonance imaging indicated the presence of vagina between the bladder and the rectum, and it showed a mass measuring 15×8 mm in the right inguinal canal as well as an oval gonadal mass with a size of 13×7 mm in the left scrotum. During surgical intervention, performed at the age of one, the right gonad was removed and biopsy of the scrotal testis was performed. Histological examination revealed dysgenetic testis in both sides. The authors emphasize the necessity of cytogenetic and endocrinological investigations of newborns with perineoscrotal hypospadia and bilateral or unilateral maldescent testes immediately after birth. Surgical removal of the dysgenetic testicular tissue located in the abdominal cavity and its histological evaluation provides separation of mixed gonadal dysgenesis, dysgenetic male pseudohermaphroditism, bilateral gonadal dysgenesis and ovotestis in the 45,X/46,XY mosaic cases. An accurate evaluation is necessary for a correct sex assignment and for surgical intervention to prevent neoplastic degeneration of the dysgenetic gonad.

Cytogenetic Investigation of Infertile Patients in Hungary: A 10-Year Retrospective Study.

Chromosome abnormalities play a crucial role in reproductive failure. The presence of numerical or structural aberrations may induce recurrent pregnancy loss or primary infertility. The main purpose of our study was to determine the types and frequency of chromosomal aberrations in infertile patients and to compare the frequency of structural aberrations to a control group. Karyotyping was performed in 1489 men and 780 women diagnosed with reproductive failure between 2010 and 2020. The control group included 869 male and 1160 female patients having cytogenetic evaluations for reasons other than infertility. Sex chromosomal aberrations were detected in 33/1489 (2.22%) infertile men and 3/780 (0.38%) infertile women. Structural abnormalities (e.g., translocation, inversion) were observed in 89/1489 (5.98%) infertile men and 58/780 (7.44%) infertile women. The control population showed structural chromosomal abnormalities in 27/869 (3.11%) men and 39/1160 (3.36%) women. There were significant differences in the prevalence of single-cell translocations between infertile individuals (males: 3.5%; females: 3.46%) and control patients (males: 0.46%; females: 0.7%). In summary, this is the first report of cytogenetic alterations in infertile patients in Hungary. The types of chromosomal abnormalities were comparable to previously published data. The prevalence of less-studied single-cell translocations was significantly higher in infertile patients than in the control population, supporting an earlier suggestion that these aberrations may be causally related to infertility.

MED13L-related intellectual disability due to paternal germinal mosaicism.

The MED13L-related intellectual disability or MRFACD syndrome (Mental retardation and distinctive facial features with or without cardiac defects; MIM # 616789) is one of the most common forms of syndromic intellectual disability with about a hundred cases reported so far. Affected individuals share overlapping features comprising intellectual disability, hypotonia, motor delay, remarkable speech delay, and a recognizable facial gestalt. De novo disruption of the MED13L gene by deletions, duplications, or sequence variants has been identified as deleterious. Siblings affected by intragenic deletion transmitted from a mosaic parent have been reported once in the literature. We now present the first case of paternal germinal mosaicism for a missense MED13L variant causing MRFACD syndrome in one of the father's children and being the likely cause of intellectual disability and facial dysmorphism in the other. As part of the Mediator complex, the MED proteins have an essential role in regulating transcription. Thirty-two subunits of the Mediator complex genes have been linked to congenital malformations that are now acknowledged as transcriptomopathies. The MRFACD syndrome has been suggested to represent a recognizable phenotype.

Deep Molecular and In Silico Protein Analysis of p53 Alteration in Myelodysplastic Neoplasia and Acute Myeloid Leukemia.

BACKGROUND: Mutation of the TP53 gene is one of the major drivers of myelodysplastic neoplasias (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MR). TP53 mutations present in these hematopoietic malignancies form a distinct molecular genetic cluster with a worse prognosis than without the alteration. However, besides well-characterized hot-spot variants, a significant proportion of TP53 alterations are of uncertain clinical significance. METHODS: To enlighten so far unknown aspects, bone-marrow samples from altogether 77 patients are analyzed retrospectively with the diagnosis of AML-MR (26 cases), MDS-IB (12 cases), and MDS-LB (39 cases) according to WHO 2022 guidelines. Next-generation sequencing results are correlated with histological, cytogenetic, and survival data. RESULTS: Twenty out of the 30 TP53 mutation types detected by NGS are not categorized in current public databases; thus, their clinical significance remained mysterious. Because of the interpretation difficulties and the absence of clinical correlations, pathogenicity is established based on in silico approaches. The 12 pathogenicity classification systems, as well as protein stability, protein-DNA, protein-protein interaction, and post-translational modification analyses are applied. We found statistically significant differences between AML/MDS groups considering p53 pathogenicity, protein structural changes, and overall survival. The largest number of abnormalities with the most severe consequences are found in AML-MR cases. CONCLUSIONS: These molecular and in silico protein data further support that MDS with increased-blast (MDS-IB) is an intermediate group between AML-MR and MDS with low-blast (MDS-LB) patients, which frequently progresses to AML and is therefore considered a pre-leukemic condition.

Helsmoortel-Van der Aa Syndrome-Cardiothoracic and Ectodermal Manifestations in Two Patients as Further Support of a Previous Observation on Phenotypic Overlap with RASopathies.

The ADNP-gene-related neurodevelopmental disorder Helsmoortel-Van der Aa syndrome is a rare syndromic-intellectual disability-an autism spectrum disorder first described by Helsmoortel and Van der Aa in 2014. Recently, a large cohort including 78 patients and their detailed phenotypes were presented by Van Dijck et al., 2019, who reported developmental delay, speech delay and autism spectrum disorder as nearly constant findings with or without variable cardiological, gastroenterological, urogenital, endocrine and neurological manifestations. Among cardiac malformations, atrial septal defect, patent ductus arteriosus, patent foramen ovale and mitral valve prolapse were the most common findings, but other unspecified defects, such as mild pulmonary valve stenosis, were also described. We present two patients with pathogenic ADNP variants and unusual cardiothoracic manifestations-Bland-White-Garland syndrome, pectus carinatum superiorly along the costochondral junctions and pectus excavatum inferiorly in one patient, and Kawasaki syndrome with pericardiac effusion, coronary artery dilatation and aneurysm in the other-who were successfully treated with intravenous immunoglobulin, corticosteroid and aspirin. Both patients had ectodermal and/or skeletal features overlapping those seen in RASopathies, supporting the observations of Alkhunaizi et al. 2018. on the clinical overlap between Helsmoortel-Van der Aa syndrome and Noonan syndrome. We observed a morphological overlap with the Noonan-like disorder with anagen hair in our patients.

Case Report: Expressive Speech Disorder in a Family as a Hallmark of 7q31 Deletion Involving the FOXP2 Gene.

Pathogenic variants of FOXP2 gene were identified first as a monogenic cause of childhood apraxia of speech (CAS), a complex disease that is associated with an impairment of the precision and consistency of movements underlying speech, due to deficits in speech motor planning and programming. FOXP2 variants are heterogenous; single nucleotide variants and small insertions/deletions, intragenic and large-scale deletions, as well as disruptions by structural chromosomal aberrations and uniparental disomy of chromosome 7 are the most common types of mutations. FOXP2-related speech and language disorders can be classified as "FOXP2-only," wherein intragenic mutations result in haploinsufficiency of the FOXP2 gene, or "FOXP2-plus" generated by structural genomic variants (i.e., translocation, microdeletion, etc.) and having more likely developmental and behavioral disturbances adjacent to speech and language impairment. The additional phenotypes are usually related to the disruption/deletion of multiple genes neighboring FOXP2 in the affected chromosomal region. We report the clinical and genetic findings in a family with four affected individuals having expressive speech impairment as the dominant symptom and additional mild dysmorphic features in three. A 7.87 Mb interstitial deletion of the 7q31.1q31.31 region was revealed by whole genome diagnostic microarray analysis in the proband. The FOXP2 gene deletion was confirmed by multiplex ligation-dependent probe amplification (MLPA), and all family members were screened by this targeted method. The FOXP2 deletion was detected in the mother and two siblings of the proband using MLPA. Higher resolution microarray was performed in all the affected individuals to refine the extent and breakpoints of the 7q31 deletion and to exclude other pathogenic copy number variants. To the best of our knowledge, there are only two family-studies reported to date with interstitial 7q31 deletion and showing the core phenotype of FOXP2 haploinsufficiency. Our study may contribute to a better understanding of the behavioral phenotype of FOXP2 disruptions and aid in the identification of such patients. We illustrate the importance of a targeted MLPA analysis suitable for the detection of FOXP2 deletion in selected cases with a specific phenotype of expressive speech disorder. The "phenotype first" and targeted diagnostic strategy can improve the diagnostic yield of speech disorders in the routine clinical practice.