Early spinal muscular atrophy treatment following newborn screening: A 20-month review of the first Italian regional experience.

OBJECTIVES: Mandatory newborn screening (NBS) for spinal muscular atrophy (SMA) was implemented for the first time in Italy at the end of 2021, allowing the identification and treatment of patients at an asymptomatic stage. METHODS: DNA samples extracted from dried blood spot (DBS) from newborns in Apulia region were analysed for SMA screening by using a real-time PCR-based assay. Infants harbouring homozygous deletion of SMN1 exon 7 confirmed by diagnostic molecular tests underwent clinical and neurophysiological assessment and received a timely treatment. RESULTS: Over the first 20 months since regional NBS introduction, four out of 42,492 (0.009%) screened children were found to carry a homozygous deletion in the exon 7 of SMN1 gene, with an annual incidence of 1:10,623. No false negatives were present. Median age at diagnosis was 7 days and median age at treatment was 20.5 days. Three of them had two copies of SMN2 and received gene therapy, while the one with three SMN2 copies was treated with nusinersen. All but one were asymptomatic at birth, showed no clinical signs of disease after a maximum follow-up of 16 months and reached motor milestones appropriate with their age. The minimum interval between diagnosis and the treatment initiation was 9 days. INTERPRETATION: The timely administration of disease-modifying therapies prevented presymptomatic subjects to develop disease symptoms. Mandatory NBS for SMA should be implemented on a national scale.

First report of whole CFTR gene duplication in a healthy newborn carrying R74W and V855I variants on the same allele.

Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder among Caucasians. The improvement of genetic techniques has allowed the identification of an increasing number of genetic variants, including large rearrangements such as duplications. We report the first case of a whole CFTR gene duplication in a healthy newborn, who had normal sweat test, also carrying R74W and V855I variants on the same allele. Familial segregation analysis and the observed frequencies of all the CFTR gene variants, revealed that R74W and V855I were probably both present in a cis arrangement on the allele also containing the duplication (i.e., in a double complex allele). Since R74W is a "variant of varying clinical consequence" its arrangement in trans with one pathogenic variant may not be sufficient to cause a classic CF disease phenotype. Moreover, its duplication could even be an advantage that could compensate for the effect of the alteration.

A Rare Case of Concurrent 2q34q36 Duplication and 2q37 Deletion in a Neonate with Syndromic Features.

Large-scale genomic structural variations can have significant clinical implications, depending on the specific altered genomic region. Briefly, 2q37 microdeletion syndrome is a prevalent subtelomeric deletion disorder characterized by variable-sized deletions. Affected patients exhibit a wide range of clinical manifestations, including short stature, facial dysmorphism, and features of autism spectrum disorder, among others. Conversely, isolated duplications of proximal chromosome 2q are rare and lack a distinct phenotype. In this report, we provide an extensive molecular analysis of a 15-day-old newborn referred for syndromic features. Our analysis reveals an 8.5 Mb microdeletion at 2q37.1, which extends to the telomere, in conjunction with an 8.6 Mb interstitial microduplication at 2q34q36.1. Our findings underscore the prominence of 2q37 terminal deletions as commonly reported genomic anomalies. We compare our patient's phenotype with previously reported cases in the literature to contribute to a more refined classification of 2q37 microdeletion syndrome and assess the potential impact of 2q34q36.1 microduplication. We also investigate multiple hypotheses to clarify the genetic mechanisms responsible for the observed genomic rearrangement.

Whole-Exome and Transcriptome Sequencing Expands the Genotype of Majewski Osteodysplastic Primordial Dwarfism Type II.

Microcephalic Osteodysplastic Primordial Dwarfism type II (MOPDII) represents the most common form of primordial dwarfism. MOPD clinical features include severe prenatal and postnatal growth retardation, postnatal severe microcephaly, hypotonia, and an increased risk for cerebrovascular disease and insulin resistance. Autosomal recessive biallelic loss-of-function genomic variants in the centrosomal pericentrin (PCNT) gene on chromosome 21q22 cause MOPDII. Over the past decade, exome sequencing (ES) and massive RNA sequencing have been effectively employed for both the discovery of novel disease genes and to expand the genotypes of well-known diseases. In this paper we report the results both the RNA sequencing and ES of three patients affected by MOPDII with the aim of exploring whether differentially expressed genes and previously uncharacterized gene variants, in addition to PCNT pathogenic variants, could be associated with the complex phenotype of this disease. We discovered a downregulation of key factors involved in growth, such as IGF1R, IGF2R, and RAF1, in all three investigated patients. Moreover, ES identified a shortlist of genes associated with deleterious, rare variants in MOPDII patients. Our results suggest that Next Generation Sequencing (NGS) technologies can be successfully applied for the molecular characterization of the complex genotypic background of MOPDII.

TNFRSF13B gene mutation in familial acute myeloid leukemia: A new piece in the complex scenario of hereditary predisposition?

TNFRSF13B mutations are widely associated with common variable immunodeficiency. TNFRSF13B was recently counted among relevant genes associated with childhood-onset of hematological malignancies; nonetheless, its role in acute myeloid leukemia (AML) remains unexplored. We report the study of a family with two cases of AML, sharing a germline TNFRSF13B mutation favoring the formation of a more stable complex with its ligand TNFSF13: a positive regulator of AML-initiating cells. Our data turn the spotlight onto the TNFRSF13B role in AML onset, inserting a new fragment into the complex scenario of a hereditary predisposition to myeloid neoplasms.

'Compressed Baby Head': A New 'Abusive Head Trauma' Entity?

BACKGROUND: Child abuse represents an important issue in the medico-legal and social context. In the last few decades, various aspects and mechanisms have been identified in child abuse case studies; however, constant research is needed in the field. With this paper, the authors will present a case of a new entity of Abusive Head Trauma that has come to the attention of medico-legal experts. DISCUSSION: The trauma analysis performed on the cranio-encephalic district of the baby revealed quite peculiar lesions that led the authors to exclude that the injuries had been solely caused by violent shaking of the baby's head, as suggested by Shaken Baby Syndrome. Instead, the authors hypothesised that another lesion mechanism had been added to this one, namely latero-lateral cranial compression. The comprehensive and exhaustive analysis of the case led the authors to present a new possible entity in child abuse trauma, namely 'Compressed Baby Head'. CONCLUSIONS: To the best of our knowledge, in the current literature, no similar clinical cases have ever been described. Thus, the case's uniqueness deserves to be brought to the attention of experts and the entire scientific community, as well as medical personnel, paediatricians, and reanimators. These professional figures are the first individuals who may encounter complex clinical cases such as the one presented in this paper; thus, they need to know how to properly manage the case and ensure protection for the abused infants and children.

Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve split-hand/foot malformation type 3.

Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1/FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigate the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1/Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice results in restructuring of the chromatin architecture. This leads to ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8-like pattern induced by AER-specific enhancers of Fgf8. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression in the developing limb. Our results reveal insights into the molecular mechanism underlying SHFM3 and provide conceptual framework for how genomic rearrangements can cause gene misexpression and disease.

Positive predictive values and outcomes for uninformative cell-free DNA tests: An Italian multicentric Cytogenetic and cytogenomic Audit of diagnOstic testing (ICARO study).

OBJECTIVES: To establish the positive predictive values (PPV) of cfDNA testing based on data from a nationwide survey of independent clinical cytogenetics laboratories. METHODS: Prenatal diagnostic test results obtained by Italian laboratories between 2013 and March 2020 were compiled for women with positive non-invasive prenatal tests (NIPT), without an NIPT result, and cases where there was sex discordancy between the NIPT and ultrasound. PPV and other summary data were reviewed. RESULTS: Diagnostic test results were collected for 1327 women with a positive NIPT. The highest PPVs were for Trisomy (T) 21 (624/671, 93%) and XYY (26/27, 96.3%), while rare autosomal trisomies (9/47, 19.1%) and recurrent microdeletions (8/55, 14.5%) had the lowest PPVs. PPVs for T21, T18, and T13 were significantly higher when diagnostic confirmation was carried out on chorionic villi (97.5%) compared to amniotic fluid (89.5%) (p < 0.001). In 19/139 (13.9%), of no result cases, a cytogenetic abnormality was detected. Follow-up genetic testing provided explanations for 3/6 cases with a fetal sex discordancy between NIPT and ultrasound. CONCLUSIONS: NIPT PPVs differ across the conditions screened and the tissues studied in diagnostic testing. This variability, issues associated with fetal sex discordancy, and no results, illustrate the importance of pre- and post-test counselling.

12q21 Interstitial Deletions: Seven New Syndromic Cases Detected by Array-CGH and Review of the Literature.

Interstitial deletions of the long arm of chromosome 12 are rare, with a dozen patients carrying a deletion in 12q21 being reported. Recently a critical region (CR) has been delimited and could be responsible for the more commonly described clinical features, such as developmental delay/intellectual disability, congenital genitourinary and brain malformations. Other, less frequent, clinical signs do not seem to be correlated to the proposed CR. We present seven new patients harboring non-recurrent deletions ranging from 1 to 18.5 Mb differentially scattered across 12q21. Alongside more common clinical signs, some patients have rarer features such as heart defects, hearing loss, hypotonia and dysmorphisms. The correlation of haploinsufficiency of genes outside the CR to specific signs contributes to our knowledge of the effect of the deletion of this gene-poor region of chromosome 12q. This work underlines the still important role of copy number variations in the diagnostic setting of syndromic patients and the positive reflection on management and family genetic counseling.

RFC1 repeat expansions: A recurrent cause of sensory and autonomic neuropathy with cough and ataxia.

BACKGROUND AND PURPOSE: Ataxia and cough are rare features in hereditary sensory and autonomic neuropathies (HSAN), a group of diseases of mostly unknown genetic cause. Biallelic repeat expansions in RFC1 are associated with cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). This study aimed to investigate the prevalence of RFC1 repeat expansions in a cohort of HSAN patients. METHODS: After unremarkable whole-exome sequencing (WES) analysis, we performed repeat-primed PCR to detect intronic RFC1 expansions in 12 HSAN families, who all presented with chronic cough. RESULTS: In these patients, 75% carried biallelic expansions of the pathogenic AAGGG motif. Compared with RFC1-/- cases, RFC1+/+ cases presented more consistently with positive sensory and autonomic symptoms. Afferent ataxia was more severe in the RFC1+/+ cohort and cerebellar ataxia was a common feature (21%). CONCLUSIONS: We demonstrate that RFC1 is a frequent cause of (WES-negative) HSAN with chronic cough and ataxia. The diagnostic yield of RFC1 repeat-primed PCR was surprisingly high, given that HSAN is genetically poorly understood. This combination of HSAN, ataxia, and chronic cough symptoms represents a new nosological entity within the neuropathy-ataxia spectrum.

Adult phenotype in Koolen-de Vries/KANSL1 haploinsufficiency syndrome.

BACKGROUND: Koolen-de Vries syndrome (KdVS) is a multisystem neurodevelopmental disorder caused by 17q21.31 deletions or mutations in KANSL1. It was mainly described in children. METHODS: A retrospective study on 9 subjects aged 19-45 years and revision of 18 literature patients, with the purpose to get insights into the phenotypic evolution with time, and into the clinical manifestations in adulthood. RESULTS: Seven patients had a 17q21.31 deletion and two a point mutation in KANSL1. All had intellectual disability, which was mild in five (56%) and moderate in four (44%). Epilepsy was diagnosed in four subjects (44%), with onset from 1 to 7 years and full remission before 9 years in 3/4 patients. Scoliosis affected seven individuals (77.7%) and it was substantially stable with age in 5/7 patients, allowing for simple daily activities. Two subjects had severely progressive scoliosis, which was surgically corrected. Overweight or true obesity did occur after puberty in six patients (67%). Behaviour abnormalities were recorded in six patients (67%). The facial phenotype slightly evolved with time to include thick eyebrows, elongated nose and pronounced pointed chin. Despite behaviour abnormalities, happy disposition and sociable attitudes were common. Half of patients had fluent language and were good at writing and reading. Rich language, although limited to single words or short sentences, and very limited or absent skills in writing and reading were observed in the remaining patients. Autonomy in daily activities and personal care was usually limited. CONCLUSIONS: Distinctive features in adult KdVS subjects include intellectual disability, overweight/obesity, behaviour abnormalities with preserved social interest, ability in language, slight worsening of the facial phenotype and no seizures.

Prenatal diagnosis of Pfeiffer syndrome and role of three-dimensional ultrasound: case report and review of literature.

Purpose/aim of the study: We report a rare case of autosomal dominant genetic syndrome "Pfeiffer", which is part of the group of acrocephalosyndactyly, with an annual incidence <1/100,000. Three forms are known. Type I is the less common form and it is characterized by moderate-severe mediofacial hypoplasia usually with normal cognitive development. Conversely, types 2 and 3 are more common and they are associated with more severe signs and complications with a more unfavorable prognosis. The type 3 form due to the presence of a cloverleaf skull distinguishes type 2.Materials and methods: Thirty-eight-year-old primigravida was referred to our center, at 28 weeks of gestation due to borderline ventriculomegaly, macrocrania, and a short femur. First trimester screening for chromosomopathies and CF-DNA was low risk; II trimester screening ultrasound showed the presence of "short femur" and macrocrania.Result: Our ultrasound evaluation, assisted by 3D ultrasound, showed cloverleaf skull, turricephaly, moderate ventriculomegaly (13 mm), hypertelorism and exophthalmos, low ear implantation, mild rhizomelia. Ultrasound depicts Pfeiffer syndrome or other acrocephalosyndactyly syndromes (Apert syndromes, Saethre-Chotzen) or other syndromic forms of craniosynostosis like Crouzon syndrome. The NGS panel for molecular analysis of genes involved in skeletal dysplasias showed the mutation of the FGFR2 gene, de novo.Conclusions: Using three-dimensional (3D) ultrasound, it is easier to distinguish rare syndromes characterized by facial dysmorphisms such as exophthalmos, mediofacial hypoplasia, and craniosynostosis.

Clinical characterization of a novel RAB39B nonstop mutation in a family with ASD and severe ID causing RAB39B downregulation and study of a Rab39b knock down mouse model.

Autism spectrum disorder (ASD) and intellectual disability (ID) often exist together in patients. The RAB39B gene has been reported to be mutated in ID patients with additional clinical features ranging from ASD, macrocephaly, seizures and/or early-onset parkinsonism. Here, we describe a novel RAB39B nonstop mutation [Xq28; c.640 T > C; p.(*214Glnext*21)] in a family with ASD, severe ID and poor motor coordination, and we assessed the pathogenicity of the mutation. A heterologous cell system and a Rab39b knockdown (KD) murine model, which mimic the nonstop mutation, were used to validate the deleterious effect of the RAB39B mutation. The mutation led to RAB39B protein instability, resulting in its increased degradation and consequent downregulation. Using a Rab39b KD mouse model, we demonstrated that the downregulation of RAB39B led to increased GluA2 lacking Ca2+-permeable AMPAR composition at the hippocampal neuronal surface and increased dendritic spine density that remained in an immature filopodia-like state. These phenotypes affected behavioural performance in a disease-specific manner. Rab39b KD mice revealed impaired social behaviour but intact social recognition. They also showed normal anxiety-like, exploratory and motivational behaviours but impaired working and associative memories. In conclusion, we found a novel RAB39B nonstop variant that segregated in a family with a clinical phenotype including ID, ASD and poor motor coordination. The pathogenicity of mutations causing the downregulation of RAB39B proteins, impacting AMPAR trafficking and dendritic spine morphogenesis, reinforced the idea that AMPAR modulation and dendritic spine assets could be considered hallmarks of neurodevelopmental disorders.

Genetic Pattern, Orthodontic and Surgical Management of Multiple Supplementary Impacted Teeth in a Rare, Cleidocranial Dysplasia Patient: A Case Report.

Background: Cleidocranial dysplasia (CCD) is a rare, autosomal dominant skeletal dysplasia with a prevalence of one per million births. The main causes of CCD are mutations in the core-binding factor alpha-1 (CBFA1) or runt-related transcription factor-2 (RUNX2), located at the 6p21 chromosomal region. RUNX2 plays important roles in osteoblast differentiation, chondrocyte proliferation and differentiation, and tooth formation. The disease is characterized by clavicular aplasia or hypoplasia, Wormian bones, delayed closure of cranial suture, brachycephalic head, maxillary deficiency, retention of primary teeth, inclusion of permanent teeth, and multiple supernumerary teeth. Materials and Methods: A 22-year-old girl suffering from cleidocranial dysplasia with short stature, narrow shoulders, craniofacial manifestations (short face, broad forehead, etc.) and dental anomalies (different lower dental elements under eruption, supernumerary and impacted multiple teeth, etc.) was examined at our service (Complex Operative Unit of Odontostomatology of Policlinico of Bari). RX Orthopantomography (OPG) and cone beam computed tomography (CBCT) were requested to better assess the position of the supernumerary teeth and their relationships with others and to evaluate the bone tissue. Results: Under eruption was probably caused by dental interferences with supernumerary teeth; hence, extractions of supernumerary upper canines and lower premolars were performed under general anaesthesia. Surgery outcome was excellent with good tissue healing and improvements in the therapeutic possibilities with future orthodontics. Conclusions: The objective of this article is to give an update about radiological, clinical, and molecular features of CCD and to alert the health team about the importance of establishing an early diagnosis and an appropriate treatment in these patients to prevent impacted teeth complications and to offer them a better quality of life.

Cytogenetic and Array-CGH Characterization of a Simple Case of Reciprocal t(3;10) Translocation Reveals a Hidden Deletion at 5q12.

Chromosome deletions, including band 5q12, have rarely been reported and have been associated with a wide range of clinical manifestations, such as postnatal growth retardation, intellectual disability, hyperactivity, nonspecific ocular defects, facial dysmorphism, and epilepsy. In this study, we describe for the first time a child with growth retardation in which we identified a balanced t(3;10) translocation by conventional cytogenetic analysis in addition to an 8.6 Mb 5q12 deletion through array-CGH. Our results show that the phenotypic abnormalities of a case that had been interpreted as "balanced" by conventional cytogenetics are mainly due to a cryptic deletion, highlighting the need for molecular investigation in subjects with an abnormal phenotype before assuming the cause is an apparently simple cytogenetic rearrangement. Finally, we identify PDE4D and PIK3R1 genes as the two major candidates responsible for the clinical features expressed in our patient.

First prenatal case of Noonan syndrome with SOS2 mutation: Implications of early diagnosis for genetic counseling.

RASopathies are a group of syndromes with partially overlapping clinical features caused by germline mutations of the RAS/MAPK signaling pathway genes. The most common disorder is Noonan syndrome (NS; MIM 163950). We report the first prenatal case of NS with SOS2 (NM_006939.4) mutation in a euploid fetus with a severe increase in nuchal translucency (NT > 12 mm). Trio-based custom next-generation sequencing detected a de novo heterozygous missense mutation in the SOS2 gene: c.800 T > A (p.Met267Lys). Owing to the marked variable expressivity of NS and the scarcity of SOS2 mutation-related NS cases reported in the literature, it is difficult to provide appropriate genetic counseling. Several issues such as the best management technique and optimal NT cutoff have been discussed. In addition, in general, the fine balance between the advantages of an early prenatal diagnosis and the challenge of determining if the detected gene variant is pathogenic and, primarily, the stress of the counselees when providing a genetic counseling with limited information on the prenatal phenotype have been discussed. A prenatal path comprising examinations and multidisciplinary counseling is essential to support couples in a shared decision-making process.

Functional evidence of mTORß splice variant involvement in the pathogenesis of congenital heart defects.

mTOR dysregulation has been described in pathological conditions, such as cardiovascular and overgrowth disorders. Here we report on the first case of a patient with a complex congenital heart disease and an interstitial duplication in the short arm of chromosome 1, encompassing part of the mTOR gene. Our results suggest that an intragenic mTOR microduplication might play a role in the pathogenesis of non-syndromic congenital heart defects (CHDs) due to an upregulation of mTOR/Rictor and consequently an increased phosphorylation of PI3K/AKT and MEK/ERK signaling pathways in patient-derived amniocytes. This is the first report which shows a causative role of intragenic mTOR microduplication in the etiology of an isolated complex CHD.

Incidence of chromosomal abnormalities in fetuses with first trimester ultrasound anomalies and a low-risk cell-free DNA test for common trisomies.

OBJECTIVE: To examine the incidence and type of chromosomal abnormalities in fetuses with first trimester ultrasound anomalies and a low-risk cfDNA test for common trisomies. METHODS: In 486 singleton pregnancies undergoing invasive testing after combined screening, a detailed first trimester ultrasound assessment was carried out and a maternal blood sample was sent for cfDNA analysis. Ultrasound and cfDNA data were analyzed in relation to fetal karyotype. RESULTS: Invasive testing demonstrated a chromosomal abnormality in 157 (32.3%) of 486 fetuses. In 348 cases with a low-risk cfDNA test for common trisomies, NT ≥ 3.5 mm and/or a major structural defect were observed in 92 (26.4%) fetuses. A chromosomal abnormality was found in 17 (18.5%; 95%CI 10.55-26.41) of these pregnancies, including 1 (1.1%) case of trisomy 21 and 16 (17.4%) fetuses with abnormalities different from common trisomies. The respective incidence in the 256 cases with a low-risk cfDNA test result and no ultrasound anomalies was 2.3% (95% CI 0.49-4.20; n = 6). CONCLUSIONS: In fetuses with first trimester ultrasound anomalies and a low-risk cfDNA result for trisomy 21, 18 and 13, diagnostic testing should be offered with the main objective to detect chromosomal abnormalities beyond common trisomies.

Bilateral Phyllodes Giant Tumor. A Case Report Analyzed by Array-CGH.

The breast phyllodes tumor is a biphasic tumor that accounts for less than of 1% of all breast neoplasms. It is classified as benign, borderline, or malignant, and can mimic benign masses. Some recurrent alterations have been identified. However, a precise molecular classification of these tumors has not yet been established. Herein, we describe a case of a 43-year-old woman that was admitted to the emergency room for a significant bleeding from the breast skin. A voluminous ulcerative mass of the left breast and multiple nodules with micro-calcifications on the right side were detected at a physical examination. A left total mastectomy and a nodulectomy of the right breast was performed. The histological diagnosis of the surgical specimens reported a bilateral giant phyllodes tumor, showing malignant features on the left and borderline characteristics associated with a fibroadenoma on the right. A further molecular analysis was carried out by an array-Comparative Genomic Hybridization (CGH) to characterize copy-number alterations. Many losses were detected in the malignant mass, involving several tumor suppressor genes. These findings could explain the malignant growth and the metastatic risk. In our study, genomic profiling by an array-CGH revealed a greater chromosomal instability in the borderline mass (40 total defects) than in the malignant (19 total defects) giant phyllodes tumor, reflecting the tumor heterogeneity. Should our results be confirmed with more sensitive and specific molecular tests (DNA sequencing and FISH analysis), they could allow a better selection of patients with adverse pathological features, thus optimizing and improving patient's management.