Mcph1, mutated in primary microcephaly, is also crucial for erythropoiesis.

Microcephaly is a common feature in inherited bone marrow failure syndromes, prompting investigations into shared pathways between neurogenesis and hematopoiesis. To understand this association, we studied the role of the microcephaly gene Mcph1 in hematological development. Our research revealed that Mcph1-knockout mice exhibited congenital macrocytic anemia due to impaired terminal erythroid differentiation during fetal development. Anemia's cause is a failure to complete cell division, evident from tetraploid erythroid progenitors with DNA content exceeding 4n. Gene expression profiling demonstrated activation of the p53 pathway in Mcph1-deficient erythroid precursors, leading to overexpression of Cdkn1a/p21, a major mediator of p53-dependent cell cycle arrest. Surprisingly, fetal brain analysis revealed hypertrophied binucleated neuroprogenitors overexpressing p21 in Mcph1-knockout mice, indicating a shared pathophysiological mechanism underlying both erythroid and neurological defects. However, inactivating p53 in Mcph1-/- mice failed to reverse anemia and microcephaly, suggesting that p53 activation in Mcph1-deficient cells resulted from their proliferation defect rather than causing it. These findings shed new light on Mcph1's function in fetal hematopoietic development, emphasizing the impact of disrupted cell division on neurogenesis and erythropoiesis - a common limiting pathway.

Expanding the molecular spectrum of pathogenic SHOC2 variants underlying Mazzanti syndrome.

We previously molecularly and clinically characterized Mazzanti syndrome, a RASopathy related to Noonan syndrome that is mostly caused by a single recurrent missense variant (c.4A > G, p.Ser2Gly) in SHOC2, which encodes a leucine-rich repeat-containing protein facilitating signal flow through the RAS-mitogen-associated protein kinase (MAPK) pathway. We also documented that the pathogenic p.Ser2Gly substitution causes upregulation of MAPK signaling and constitutive targeting of SHOC2 to the plasma membrane due to the introduction of an N-myristoylation recognition motif. The almost invariant occurrence of the pathogenic c.4A > G missense change in SHOC2 is mirrored by a relatively homogeneous clinical phenotype of Mazzanti syndrome. Here, we provide new data on the clinical spectrum and molecular diversity of this disorder and functionally characterize new pathogenic variants. The clinical phenotype of six unrelated individuals carrying novel disease-causing SHOC2 variants is delineated, and public and newly collected clinical data are utilized to profile the disorder. In silico, in vitro and in vivo characterization of the newly identified variants provides evidence that the consequences of these missense changes on SHOC2 functional behavior differ from what had been observed for the canonical p.Ser2Gly change but converge toward an enhanced activation of the RAS-MAPK pathway. Our findings expand the molecular spectrum of pathogenic SHOC2 variants, provide a more accurate picture of the phenotypic expression associated with variants in this gene and definitively establish a gain-of-function behavior as the mechanism of disease.

Dermatological manifestations in Costello syndrome: A prospective multicentric study of 31 HRAS-positive variant patients.

BACKGROUND: Data on dermatological manifestations of Costello syndrome (CS) remain heterogeneous and lack in validated description. OBJECTIVES: To describe the dermatological manifestations of CS; compare them with the literature findings; assess those discriminating CS from other RASopathies, including cardiofaciocutaneous syndrome (CFCS) and the main types of Noonan syndrome (NS); and test for dermatological phenotype-genotype correlations. METHODS: We performed a 10-year, large, prospective, multicentric, collaborative dermatological and genetic study. RESULTS: Thirty-one patients were enrolled. Hair abnormalities were ubiquitous, including wavy or curly hair and excessive eyebrows, respectively in 68% and 56%. Acral excessive skin (AES), papillomas and keratotic papules (PKP), acanthosis nigricans (AN), palmoplantar hyperkeratosis (PPHK) and 'cobblestone' papillomatous papules of the upper lip (CPPUL), were noted respectively in 84%, 61%, 65%, 55% and 32%. Excessive eyebrows, PKP, AN, CCPUL and AES best differentiated CS from CFCS and NS. Multiple melanocytic naevi (>50) may constitute a new marker of attenuated CS associated with intragenic duplication in HRAS. Oral acitretin may be highly beneficial for therapeutic management of PPHK. No significant dermatological phenotype-genotype correlation was determined between patients with and without HRAS c.34G>A (p.G12S). CONCLUSIONS AND RELEVANCE: This validated phenotypic characterization of a large number of patients with CS will allow future researchers to make a positive diagnosis, and to differentiate CS from CFCS and NS.

Retrotransposon insertion as a novel mutational cause of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder resulting from biallelic alterations of the SMN1 gene: deletion, gene conversion or, in rare cases, intragenic variants. The disease severity is mainly influenced by the copy number of SMN2, a nearly identical gene, which produces only low amounts of full-length (FL) mRNA. Here we describe the first example of retrotransposon insertion as a pathogenic SMN1 mutational event. The 50-year-old patient is clinically affected by SMA type III with a diagnostic odyssey spanning nearly 30 years. Despite a mild disease course, he carries a single SMN2 copy. Using Exome Sequencing and Sanger sequencing, we characterized a SINE-VNTR-Alu (SVA) type F retrotransposon inserted in SMN1 intron 7. Using RT-PCR and RNASeq experiments on lymphoblastoid cell lines, we documented the dramatic decrease of FL transcript production in the patient compared to subjects with the same SMN1 and SMN2 copy number, thus validating the pathogenicity of this SVA insertion. We described the mutant FL-SMN1-SVA transcript characterized by exon extension and showed that it is subject to degradation by nonsense-mediated mRNA decay. The stability of the SMN-SVA protein may explain the mild course of the disease. This observation exemplifies the role of retrotransposons in human genetic disorders.

Pediatric Tumors and Developmental Anomalies: A French Nationwide Cohort Study.

OBJECTIVE: To assess the associations between congenital abnormalities and pediatric malignancies and evaluate the potential underlying molecular basis by collecting information on pediatric patients with cancer and congenital abnormalities. STUDY DESIGN: Tumeur Et Développement is a national, prospective, and retrospective multicenter study recording data of children with cancer and congenital abnormalities. When feasible, blood and tumoral samples are collected for virtual biobanking. RESULTS: From June 2013 to December 2019, 679 associations between pediatric cancers and congenital abnormalities were recorded. The most represented cancers were central nervous system tumors (n = 139; 20%), leukemia and myelodysplastic syndromes (n = 123; 18.1%), and renal tumors (n = 101; 15%). Congenital abnormalities were not related to any known genetic disorder in 66.5% of cases. In this group, the most common anomaly was intellectual disability (22.3%), followed by musculoskeletal (14.2%) and genitourinary anomalies (12.4%). Intellectual disability was mostly associated with hematologic malignancies. Embryonic tumors (neuroblastoma, Wilms tumor, and rhabdomyosarcoma) were associated with consistent abnormalities, sometimes with a close anatomical neighborhood between the abnormality and the neoplasm. CONCLUSIONS: In the first Tumeur Et Développement analysis, 3 major themes have been identified: (1) germline mutations with or without known cancer predisposition, (2) postzygotic events responsible for genomic mosaicism, (3) coincidental associations. New pathways involved in cancer development need to be investigated to improve our understanding of childhood cancers.

Germline bi-allelic SH2B3/LNK alteration predisposes to a neonatal juvenile myelomonocytic leukemia-like disorder.

Juvenile myelomonocytic leukemia (JMML) is a rare, generally aggressive myeloproliferative neoplasm affecting young children. It is characterized by granulomonocytic expansion, with monocytosis infiltrating peripheral tissues. JMML is initiated by mutations upregulating RAS signaling. Approximately 10% of cases remain without an identified driver event. Exome sequencing of 2 unrelated cases of familial JMML of unknown genetics and analysis of the French JMML cohort identified 11 patients with variants in SH2B3, encoding LNK, a negative regulator of the JAK-STAT pathway. All variants were absent from healthy population databases, and mutation spectrum was consistent with a loss of function of the LNK protein. A stoploss variant was shown to affect both protein synthesis and stability. The other variants were either truncating or missense, the latter affecting the SH2 domain that interacts with activated JAK. Of the 11 patients, 8 from 5 families inherited pathogenic bi-allelic SH2B3 germline variants from their unaffected heterozygous parents. These children represent half of the cases with no identified causal mutation in the French cohort. They displayed typical clinical and hematological JMML features with neonatal onset and marked thrombocytopenia. They were characterized by absence of additional genetic alterations and a hypomethylated DNA profile with fetal characteristics. All patients showed partial or complete spontaneous clinical resolution. However, progression to thrombocythemia and immunity-related pathologies may be of concern later in life. Bi-allelic SH2B3 germline mutations thus define a new condition predisposing to a JMML-like disorder, suggesting that the JAK pathway deregulation is capable of initiating JMML, and opening new therapeutic options.

Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome.

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.

De novo NUF2 variant in a novel inherited bone marrow failure syndrome including microcephaly and renal hypoplasia.

In a patient with severe microcephaly, congenital bone marrow failure, growth retardation, and renal hypoplasia, we identified a likely pathogenic variant in NUF2 that impairs the cell's ability to properly complete mitosis. Interestingly, these clinical features as well as the observed cellular alterations are highly reminiscent of what is reported in Fanconi Anaemia supporting a unifying causal role of the variant in the disease. This case provides the first evidence that a kinetochore defect, previously associated with microcephaly, can be responsible for an inherited bone marrow failure syndrome, highlighting the unique pathological link between neurogenesis and haematopoiesis.

T-Cell Acute Lymphoblastic Leukemia in a Young Adult With Thrombocytopenia-absent Radius Syndrome: A Case Report and Review of the Literature.

Thrombocytopenia-absent radius (TAR) syndrome is a rare inherited bone marrow failure syndrome not generally associated with acute leukemia. The authors report a case of T-cell acute lymphoblastic leukemia in an adult female individual newly diagnosed with TAR syndrome. A 347-kb microdeletion of chromosome 1q21.1 involving the RBM8A gene was detected within a gain of whole chromosome 1. Next-generation sequencing on fibroblasts confirmed germline heterozygous deletion of RBM8A but on the other allele, noncoding low-frequency regulatory single-nucleotide polymorphism of RBM8A (rs139428292; rs201779890) were not found. The tolerance of the treatment was unusual and mostly marked by a slow hematopoietic recovery leading to a 6-month delay at the beginning of the maintenance phase. Only 5 cases of acute leukemia were reported in patients with TAR syndrome in the literature: 4 acute myeloid leukemia and one B-cell acute lymphoblastic leukemia. This is the first report of T-cell acute lymphoid leukemia occurring in the context of TAR syndrome.

Digenic inheritance of human primary microcephaly delineates centrosomal and non-centrosomal pathways.

Primary microcephaly (PM) is characterized by a small head since birth and is vastly heterogeneous both genetically and phenotypically. While most cases are monogenic, genetic interactions between Aspm and Wdr62 have recently been described in a mouse model of PM. Here, we used two complementary, holistic in vivo approaches: high throughput DNA sequencing of multiple PM genes in human patients with PM, and genome-edited zebrafish modeling for the digenic inheritance of PM. Exomes of patients with PM showed a significant burden of variants in 75 PM genes, that persisted after removing monogenic causes of PM (e.g., biallelic pathogenic variants in CEP152). This observation was replicated in an independent cohort of patients with PM, where a PM gene panel showed in addition that the burden was carried by six centrosomal genes. Allelic frequencies were consistent with digenic inheritance. In zebrafish, non-centrosomal gene casc5 -/- produced a severe PM phenotype, that was not modified by centrosomal genes aspm or wdr62 invalidation. A digenic, quadriallelic PM phenotype was produced by aspm and wdr62. Our observations provide strong evidence for digenic inheritance of human PM, involving centrosomal genes. Absence of genetic interaction between casc5 and aspm or wdr62 further delineates centrosomal and non-centrosomal pathways in PM.

Hypophosphatemic rickets: A rare complication of congenital melanocytic nevus syndrome.

We report the case of a child who presented with a giant melanocytic nevus with numerous satellite nevi at birth and developed hypophosphatemic rickets due to excessive secretion of the FGF23 hormone. A NRAS c.182A>G (Q61R) mutation was identified in the lesional skin. The functional outcome was favorable with medical treatment.

Cherubism as a systemic skeletal disease: evidence from an aggressive case.

BACKGROUND: Cherubism is a rare autosomal dominant genetic condition caused by mutations in the SH3BP2 gene. This disease is characterized by osteolysis of the jaws, with the bone replaced by soft tissue rich in fibroblasts and multinuclear giant cells. SH3BP2 is a ubiquitous adaptor protein yet the consequences of SH3BP2 mutation have so far been described as impacting only face. Cherubism mouse models have been generated and unlike human patients, the knock-in mice exhibit systemic bone loss together with a systemic inflammation. CASE PRESENTATION: In light of these observations, we decided to search for a systemic cherubism phenotype in a 6-year-old girl with an aggressive cherubism. We report here the first case of cherubism with systemic manifestations. Bone densitometry showed low overall bone density (total body Z-score = - 4.6 SD). Several markers of bone remodelling (CTx, BALP, P1NP) as well as inflammation (TNFα and IL-1) were elevated. A causative second-site mutation in other genes known to influence bone density was ruled out by sequencing a panel of such genes. CONCLUSIONS: If this systemic skeletal cherubism phenotype should be confirmed, it would simplify the treatment of severe cherubism patients and allay reservations about applying a systemic treatment such as those recently published (tacrolimus or imatinib) to a disease heretofore believed to be localised to the jaws.

Autosomal recessive primary microcephaly due to ASPM mutations: An update.

Autosomal recessive microcephaly or microcephaly primary hereditary (MCPH) is a genetically heterogeneous neurodevelopmental disorder characterized by a reduction in brain volume, indirectly measured by an occipitofrontal circumference (OFC) 2 standard deviations or more below the age- and sex-matched mean (-2SD) at birth and -3SD after 6 months, and leading to intellectual disability of variable severity. The abnormal spindle-like microcephaly gene (ASPM), the human ortholog of the Drosophila melanogaster "abnormal spindle" gene (asp), encodes ASPM, a protein localized at the centrosome of apical neuroprogenitor cells and involved in spindle pole positioning during neurogenesis. Loss-of-function mutations in ASPM cause MCPH5, which affects the majority of all MCPH patients worldwide. Here, we report 47 unpublished patients from 39 families carrying 28 new ASPM mutations, and conduct an exhaustive review of the molecular, clinical, neuroradiological, and neuropsychological features of the 282 families previously reported (with 161 distinct ASPM mutations). Furthermore, we show that ASPM-related microcephaly is not systematically associated with intellectual deficiency and discuss the association between the structural brain defects (strong reduction in cortical volume and surface area) that modify the cortical map of these patients and their cognitive abilities.

Phosphoglycerate dehydrogenase (PHGDH) deficiency without epilepsy mimicking primary microcephaly.

Phosphoglycerate dehydrogenase (PHGDH) deficiency (OMIM 256520) is a rare autosomal recessive disorder of serine synthesis, with mostly severe congenital microcephaly, caused by mutations in the PHGDH gene. Fourteen patients reported to date show severe, early onset, drug resistant epilepsy. In a cohort of patients referred for primary microcephaly, compound heterozygosity for two unreported variants in PHGDG was identified by exome sequencing in a pair of sibs who died aged 4.5 months and 4.5 years. They had severe neurological involvement with congenital microcephaly, disorganized EEG, and progressive spasticity, but never had seizures. Exome usage in clinical practice is likely to lead to an expansion of the clinical spectrum of known disorders.

Partitioning for Easy Multiplexing: A Versatile Droplet PCR Application for Clone Monitoring in Tumors.

Clinical genome-wide next-generation sequencing (NGS) has brought new challenges to genetic laboratories. The identification of numerous patient-specific variants that may require to be screened for on multiple other samples poses an issue when striving for time and cost-effectiveness. Here, we propose d-multiSeq, a straightforward method utilizing the advantages of droplet PCR for multiplexing combined with amplicon-based NGS. By comparing d-multiSeq with a standard multiplex amplicon-based NGS, it was shown that partitioning prevents the amplification competition seen when multiplexing and leads to a homogeneous representation of each target in the total read count for up to a 40-target multiplex without the need for prior adjustment. Variant allele frequency was reliably evaluated with a sensitivity of 97.6% for variant allele frequency up to 1%. The applicability of d-multiSeq was also tested on cell-free DNA with the successful amplification of an eight-target multiplex panel. Preliminary application of the technique to assess the clonal evolution in a childhood leukemia harboring high interpatient variability in its somatic variants is shown. d-multiSeq represents a turnkey solution for analyzing large sets of patient-specific variants on low DNA amounts and cell-free DNA.

Early treatment of neonatal diabetes with oral glibenclamide in an extremely preterm infant.

Early treatment of neonatal diabetes with sulfonylureas has been proven to produce marked improvements of neurodevelopment, beside the demonstrated efficacy on glycemic control. Several barriers still prevent an early treatment in preterm babies including the limited availability of suitable galenic form of glibenclamide. We adopted oral glibenclamide suspension (Amglidia) for the early treatment of neonatal diabetes due to an homozygous variant of KCNJ11 gene c.10C>T [p.Arg4Cys] in an extremely preterm infant born at 26 + 2 weeks' of gestational age. After ~6 weeks of insulin treatment with a low glucose intake (4.5 g/kg/day), the infant was switched to Amglidia 6 mg/ml diluted in maternal milk, via nasogastric tube (0.2 mg/kg/day) progressively reduced to 0.01 mg/kg/day (after ~3 months). While on glibenclamide, the patient exhibited a mean daily growth of 11 g/kg/day. The treatment was suspended at month 6 of birth (weight 4.9 kg [5th-10th centile], M3 of c.a.) for normalization of glucose profile. During the treatment, the patient exhibited a stable glucose profile within the range of 4-8 mmol/L in the absence of hypo or hyperglycemic episodes with 2-3 blood glucose tests per day. The patient was diagnosed with retinopathy of prematurity Stade II in Zone II without plus disease at 32 weeks, with progressive regression and complete retinal vascularization at 6 months of birth. Amglidia could be regarded as the specific treatment for neonatal diabetes even in preterm babies due to its beneficial effect on the metabolic and neurodevelopmental side.

Variants of SOS2 are a rare cause of Noonan syndrome with particular predisposition for lymphatic complications.

RASopathies are caused by variants in genes encoding components or modulators of the RAS/MAPK signaling pathway. Noonan syndrome is the most common entity among this group of disorders and is characterized by heart defects, short stature, variable developmental delay, and typical facial features. Heterozygous variants in SOS2, encoding a guanine nucleotide exchange factor for RAS, have recently been identified in patients with Noonan syndrome. The number of published cases with SOS2-related Noonan syndrome is still limited and little is known about genotype-phenotype correlations. We collected previously unpublished clinical and genotype data from 17 individuals carrying a disease-causing SOS2 variant. Most individuals had one of the previously reported dominant pathogenic variants; only four had novel changes at the established hotspots for variants that affect protein function. The overall phenotype of the 17 patients fits well into the spectrum of Noonan syndrome and is most similar to the phenotype observed in patients with SOS1-related Noonan syndrome, with ectodermal anomalies as common features and short stature and learning disabilities as relatively infrequent findings compared to the average Noonan syndrome phenotype. The spectrum of heart defects in SOS2-related Noonan syndrome was consistent with the known spectrum of cardiac anomalies in RASopathies, but no specific heart defect was particularly predominating. Notably, lymphatic anomalies were extraordinarily frequent, affecting more than half of the patients. We therefore conclude that SOS2-related Noonan syndrome is associated with a particularly high risk of lymphatic complications that may have a significant impact on morbidity and quality of life.