Corrigendum: Newborn screening for X-linked adrenoleukodystrophy in Italy: diagnostic algorithm and disease monitoring.

[This corrects the article DOI: 10.3389/fneur.2022.1072256.].

Comparison of first-tier whole-exome sequencing with a multi-step traditional approach for diagnosing paediatric outpatients: An Italian prospective study.

BACKGROUND: The recent guidelines suggest the use of genome-wide analyses, such as whole exome sequencing (WES), at the beginning of the diagnostic approach for cases with suspected genetic conditions. However, in many realities it still provides for the execution of a multi-step pathway, thus requiring several genetic tests to end the so-called 'diagnostic odyssey'. METHODS: We reported the results of GENE Project (Genomic analysis Evaluation NEtwork): a multicentre prospective cohort study on 125 paediatric outpatients with a suspected genetic disease in which we performed first-tier trio-WES, including exome-based copy number variation analysis, in parallel to a 'traditional approach' of two/three sequential genetic tests. RESULTS: First-tier trio-WES detected a conclusive diagnosis in 41.6% of patients, way above what was found with routine genetic testing (25%), with a time-to-result of about 50 days. Notably, the study showed that 44% of WES-reached diagnoses would be missed with the traditional approach. The diagnostic rate (DR) of the two approaches varied in relation to the phenotypic class of referral and to the proportion of cases with a defined diagnostic suspect, proving the major difference for neurodevelopmental disorders. Moreover, trio-WES analysis detected variants in candidate genes of unknown significance (EPHA4, DTNA, SYNCRIP, NCOR1, TFDP1, SPRED3, EDA2R, PHF12, PPP1R12A, WDR91, CDC42BPG, CSNK1D, EIF3H, TMEM63B, RIPPLY3) in 19.4% of undiagnosed cases. CONCLUSION: Our findings represent real-practice evidence of how first-tier genome-wide sequencing tests significantly improve the DR for paediatric outpatients with a suspected underlying genetic aetiology, thereby allowing a time-saving setting of the correct management, follow-up and family planning.

Cobblestone-like brain malformation with a new bi-allelic ADGRG1 (GPR-56) mutation: Fetal imaging-pathology correlation.

BACKGROUND AND PURPOSE: Autosomal recessive cobblestone-like cortical malformation of the brain, with no eye or muscle involvement, has been reported in patients with biallelic mutations in ADGRG1 (formerly GPR56) and in other brain surface defects (eg, variants in COL3A1). We reported the intra-uterine brain MRI (iu-MRI), post-mortem MRI (pm-MRI), and neuropathology findings of a new ADGRG1 mutation in a fetus at early gestation. Imaging findings were compared with those of the sibling harboring the same mutation, to provide insights about the evolving morphology of such malformation. METHODS: A 21-week fetus underwent iu-MRI for a suspected cortical anomaly on ultrasound. After the MRI results, the termination of the pregnancy was carried out. A pm-MRI scan and autopsy were performed. A neuropathology-imaging correlation was achieved. The 5-year old sibling affected by developmental impairment also underwent a brain MRI. Both subjects underwent a genetic investigation. RESULTS: Two patterns of abnormality of the cerebral surface were identified on both fetal MRI: one at the vertex resembling a cobblestone-cortex due to neuronal overmigration into the subarchnoid space and the other in the occipital areas resembling polymicrogyria. These details closely matched the neuropathology findings. MRI findings of the sibling consisted of typical ADGRG1/GPR56-related brain findings showing a polymicrogyric-like cortex, also reported as bilateral frontal-parietal polymicrogyria. A flattened pons and small cerebellar vermis were present in both cases. Genetic testing demonstrated a novel homozygous variant c.1484T>C in the c gene in both cases. CONCLUSION: Our findings provide further evidence of the overlap of ADGRG1/GPR56-related brain dysgenesis with cobblestone-like cortical malformation of the brain.

Menkes disease complicated by concurrent ACY1 deficiency: A case report.

Introduction: Menkes disease is an X-linked recessive condition caused by mutations in the ATP7A gene, which leads to severe copper deficiency. Aminoacylase-1 deficiency is a rare inborn error of metabolism caused by homozygous or compound heterozygous variant in the ACY1 gene, characterized by increased urinary excretion of specific N-acetyl amino acids. Case presentation: We report an infant with neurological findings such as seizures, neurodevelopmental delay and hypotonia. Metabolic screening showed low serum copper and ceruloplasmin, and increased urinary excretion of several N-acetylated amino acids. Whole-exome sequencing analysis (WES) revealed the novel de novo variant c.3642_3649dup (p.Ala1217Aspfs*2) in the ATP7A gene, leading to a diagnosis of Menkes disease, and the simultaneous presence of the homozygous ACY1 variant c.1057C>T (p.Arg353Cys) causative of Aminoacylase-1 deficiency. Conclusion: Our patient had two rare conditions with different treatment courses but overlapping clinical features. The identified novel ATP7A mutation associated with Menkes disease expands the ATP7A gene spectrum.

Expanding the Molecular Spectrum of ANKRD11 Gene Defects in 33 Patients with a Clinical Presentation of KBG Syndrome.

KBG syndrome (KBGS) is a neurodevelopmental disorder caused by the Ankyrin Repeat Domain 11 (ANKRD11) haploinsufficiency. Here, we report the molecular investigations performed on a cohort of 33 individuals with KBGS clinical suspicion. By using a multi-testing genomic approach, including gene sequencing, Chromosome Microarray Analysis (CMA), and RT-qPCR gene expression assay, we searched for pathogenic alterations in ANKRD11. A molecular diagnosis was obtained in 22 out of 33 patients (67%). ANKRD11 sequencing disclosed pathogenic or likely pathogenic variants in 18 out of 33 patients. CMA identified one full and one terminal ANKRD11 pathogenic deletions, and one partial duplication and one intronic microdeletion, with both possibly being pathogenic. The pathogenic effect was established by RT-qPCR, which confirmed ANKRD11 haploinsufficiency only for the three deletions. Moreover, RT-qPCR applied to six molecularly unsolved KBGS patients identified gene downregulation in a clinically typical patient with previous negative tests, and further molecular investigations revealed a cryptic deletion involving the gene promoter. In conclusion, ANKRD11 pathogenic variants could also involve the regulatory regions of the gene. Moreover, the application of a multi-test approach along with the innovative use of RT-qPCR improved the diagnostic yield in KBGS suspected patients.

Progressive Clinical and Neuroradiological Findings in a Child with BCL11B Missense Mutation: Expanding the Phenotypic Spectrum of Related Disorder.

We report a patient affected by BCL11B-related disorder, providing the first extensive demonstration of clinical and neuroradiological progressive course of the disease, with possible implications on the way it is studied and followed-up. Never described clinical aspects such as toes abnormalities and hypospadias widen the range of dysmorphisms associated with this condition. Our data suggest that BCL11B mutations may be implicated not only in impaired morphogenesis and hematopoiesis but also in progressive central nervous system damage, which remains to be further investigated and clarified.

Newborn screening for X-linked adrenoleukodystrophy in Italy: Diagnostic algorithm and disease monitoring.

Introduction: X-linked adrenoleukodystrophy (X-ALD) is the most common inherited peroxisomal disorder caused by variants in the ABCD1 gene. The main phenotypes observed in men with X-ALD are primary adrenal insufficiency, adrenomyeloneuropathy, and cerebral ALD (cALD). Cerebral ALD consists of a demyelinating progressive cerebral white matter (WM) disease associated with rapid clinical decline and is fatal if left untreated. Hematopoietic stem cell transplantation is the standard treatment for cALD as it stabilizes WM degeneration when performed early in the disease. For this reason, early diagnosis is crucial, and several countries have already implemented their newborn screening programs (NBS) with the assessment of C26:0-lysophosphatidylcholine (C26:0-LPC) values as screening for X-ALD. Methods: In June 2021, an Italian group in Lombardy launched a pilot study for the implementation of X-ALD in the Italian NBS program. A three-tiered approach was adopted, and it involved quantifying the values of C26:0-LPC and other metabolites in dried blood spots with FIA-MS/MS first, followed by the more specific ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique and, finally, the genetic confirmation via focused NGS. Discussion: Genetically confirmed patients are set to undergo a follow-up protocol and are periodically evaluated to promptly start a specific treatment if and when the first signs of brain damage appear, as suggested by international guidelines. A specific disease monitoring protocol has been created based on literature data and personal direct experience. Conclusion: The primary aim of this study was to develop a model able to improve the early diagnosis and subsequent follow-up and timely treatment of X-ALD. Ethics: The study was approved by the local ethics committee. The research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

Autosomal Dominant Hypophosphatemic Rickets: A Case Report and Review of the Literature.

Autosomal dominant hypophosphatemic rickets (ADHR) is an extremely rare form of genetic rickets caused by mutations in the fibroblast growth factor 23 gene. ADHR is characterized by hypophosphatemia secondary to isolated renal phosphate wasting. Only a few cases of ADHR have been reported in the literature to date. We describe the case of a 17-month-old girl who presented with severe failure to thrive (length: -4.08 standard deviation (SD), weight: -2.2 SD) and hypotonia. Hypophosphatemia, decreased tubular phosphate reabsorption (69%), and rachitic lesions were found. Genetic analysis showed the heterozygous variant c.536G>A (NM_020638.3:c.536G>A) in exon 3 of the FGF23 gene, leading to the diagnosis of ADHR. She was treated with phosphate salts and oral alfacalcidol. After 4 years of treatment, at 5 years of age, the patient's ADHR resolved spontaneously. Considering the lack of knowledge regarding ADHR, we reviewed the literature to describe the features of this rare and poorly understood disease. Eleven ADHR pediatric cases have been described thus far, with cases tending to be more common in females than males. Similar to the general population, two groups of patients with ADHR can be described depending on the mutations present: patients with an R179 and R176 mutation have early-onset of disease and higher frequency of rickets, and a milder and late-onset of disease, respectively. Symptoms and disease severity may fluctuate. Spontaneous remission may occur during the pediatric age.

Electroclinical features of MEF2C haploinsufficiency-related epilepsy: A multicenter European study.

PURPOSE: Epilepsy is a main manifestation in the autosomal dominant mental retardation syndrome caused by heterozygous variants in MEF2C. We aimed to delineate the electro-clinical features and refine the genotype-phenotype correlations in patients with MEF2C haploinsufficiency. METHODS: We thoroughly investigated 25 patients with genetically confirmed MEF2C-syndrome across 12 different European Genetics and Epilepsy Centers, focusing on the epileptic phenotype. Clinical features (seizure types, onset, evolution, and response to therapy), EEG recordings during waking/sleep, and neuroimaging findings were analyzed. We also performed a detailed literature review using the terms "MEF2C", "seizures", and "epilepsy". RESULTS: Epilepsy was diagnosed in 19 out of 25 (~80%) subjects, with age at onset <30 months. Ten individuals (40%) presented with febrile seizures and myoclonic seizures occurred in ~50% of patients. Epileptiform abnormalities were observed in 20/25 patients (80%) and hypoplasia/partial agenesis of the corpus callosum was detected in 12/25 patients (~50%). Nine patients harbored a 5q14.3 deletion encompassing MEF2C and at least one other gene. In 7 out of 10 patients with myoclonic seizures, MIR9-2 and LINC00461 were also deleted, whereas ADGRV1 was involved in 3/4 patients with spasms. CONCLUSION: The epileptic phenotype of MEF2C-syndrome is variable. Febrile and myoclonic seizures are the most frequent, usually associated with a slowing of the background activity and irregular diffuse discharges of frontally dominant, symmetric or asymmetric, slow theta waves with interposed spike-and-waves complexes. The haploinsufficiency of ADGRV1, MIR9-2, and LINC00461 likely contributes to myoclonic seizures and spasms in patients with MEF2C syndrome.

Cerebellar dysplasia related to PIK3CA mutation: a three-case series.

The term PROS (PIK3CA-Related Overgrowth Spectrum) indicates a wide spectrum of overgrowth disorders related to somatic mutations in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) pathway. We present three cases with PIK3CA mutation and clinical characteristics encompassing MCAP (megalencephaly-capillary malformation) condition but lacking all criteria to a certain diagnosis, most of all showing prevalent and peculiar involvement of cerebellar structures at MRI (magnetic resonance imaging) mainly consisting in cortical rim thickening and abnormal orientation of folia axis. These cases expand the spectrum of intracranial MRI features in PIK3CA disorders.

CD10 and CD34 as markers in vascular malformations with PIK3CA and TEK mutations.

AIMS: Vascular malformations (vMs) encompass a wide range of diseases often associated with somatic or, more rarely, germinal genetic mutations. A mutation in the PIK3Ca/mTOR pathway is more often involved in various vMs. CD10 and CD34 are cellular markers that may play a role in mesenchymal differentiation and proliferation. The aim of our study was to find a possible link between the immunohistochemical expression of CD10 and CD34 in vMs and their relationship with mutations in the PIK3CA/mTOR signaling pathway. METHODS AND RESULTS: Our study on 58 samples of vMs showed that in endothelial cells, CD10 was significantly expressed in PIK3CA-mutated samples compared with samples without any mutation (p < 0.05), especially and even more consistently when compared with samples with mutation in other pathways (p < 0.0001). Conversely, in the same PIK3CA-mutated samples, CD34 expression in endothelial cells was significantly reduced compared with samples either without any mutation or mutations in other pathways (p < 0.05 and p < 0.0005). Compared with samples with mutations in other pathways, a significant overexpression of endothelial CD10 was also found in samples with TEK/TIE2 mutation, a gene linked to the PIK3CA/mTOR pathway (p < 0.01). However, CD34 expression was not altered. In samples with PIK3CA mutation, the CD10 expression was significantly increased in the stroma compared with samples with TEK/TIE2 gene or other gene mutations (p < 0.05). CONCLUSION: Therefore, the CD10 and CD34 immunohistochemical profile could suggest/support the presence of mutations in the PIK3CA/mTOR pathway in samples of vMs.

Natural history of non-lethal Raine syndrome during childhood.

BACKGROUND: Raine syndrome (RS) is a rare autosomal recessive disorder caused by biallelic loss-of-function mutations of FAM20C. The most common clinical features are microcephaly, exophthalmos, hypoplastic nose and severe midface hypoplasia, leading to choanal atresia. The radiological findings include generalized osteosclerosis and brain calcifications. RS is usually lethal during the neonatal period due to severe respiratory distress. However, there exists a non-lethal RS form, the phenotype of which is extremely heterogeneous. There is paucity of data about clinical course and life expectancy of these patients. RESULTS: This is the first description of follow-up features of non-lethal RS patients. Moreover, we present three unpublished cases. There are five Asian and two Arab patients. All were born to consanguineous parents. The most common neonatal comorbidity was respiratory distress secondary to choanal atresia. A variable degree of neurodevelopmental delay was seen in the majority of our cases and seizures and hearing or vision involvement were also frequent. Neurological and orthopedic issues were the most frequent complications seen at follow-up in our group. Persistent hypophosphatemic rickets was the most striking endocrinological manifestation, which was scarcely responsive to therapy with phosphate salts and alfacalcidol. Life expectancy of our patients goes beyond childhood, with the oldest of those described being 18 years old at present. CONCLUSIONS: Manifestations of RS in those surviving the neonatal period are being increasingly recognized. Our study supports previous findings and provides clinical and biochemical observations and data from longer follow up. Finally, we propose multidisciplinary follow up for patients with non-lethal RS.

Currarino syndrome and microcephaly due to a rare 7q36.2 microdeletion: a case report.

BACKGROUND: Currarino syndrome is a rare condition characterized by presacral mass, anorectal malformation and sacral dysgenesis. CASE PRESENTATION: We report the case of a child that presented chronic constipation, encopresis and mycrocephaly. The characteristics were initially compatible with a case of functional constipation and a therapy with polyethylene glycol was prescribed. After a year, because of poor response, a plain abdominal X-ray was performed, detecting sacrum abnormalities. Finally, a CGH-array analysis was performed and a form of Currarino Syndrome caused by a rare 7q36 microdeletion, was diagnosed. CONCLUSION: Occult spinal dysraphism should be suspected in case of poor polyethylene glycol responder constipation, even when evident sacral abnormalities on the physical examination are not detected.

Moebius syndrome: clinical features, diagnosis, management and early intervention.

BACKGROUND: Moebius syndrome (MBS) is rare disease characterized by nonprogressive congenital uni- or bi-lateral facial (i. e. VII cranial nerve) and abducens (i. e. VI cranial nerve) palsy. Although the neurological and ophthalmological findings are quite well-known, data concerning the attendant functional difficulties and their changes over time are seldom addressed. In this study we attempt to estimate the prevalence of clinical and functional data in an Italian cohort affected by MBS. METHODS: The study included 50 children, 21 males and 29 females, aged 1 month to 14 years. The patients entered into a multidisciplinary diagnostic and follow-up protocol that had the specific purpose of detecting clinical and developmental deficits related to MBS. RESULTS: Involvement of the VII cranial nerve (total/partial, bilateral or unilateral) was present in 96 % of patients, and of the VI nerve in 85 %. Two patients were without impairment of the VII nerve and seven patients had no involvement of the VI nerve and were thus classified as Moebius-like because of the involvement of other CNs. Additional affected CNs were numbers III-IV in 16 %, V in 11 %, VIII and X each in 8 %, the XI in 6 %, the IX, most often partially, in 22 %, and the XII in 48 % of cases. Their development was characterized by global delay at one year of age, motor, emotional and speech difficulties at two years of age, a trend toward normalization at three years of age but with weakness in hand-eye coordination, and achieving average results at five years of age. Overall 90 % of children had a normal developmental quotient whereas only 10 % manifested cognitive deficits. CONCLUSION: Early rehabilitation may enhance the recovery of normal function, particularly in vulnerable areas of development. It is possible that early intervention that integrates sensory and visual information with emotional difficulties can improve the prognosis of the child with MBS.

Rare interstitial deletion of chromosome 2p11.2p12. Report of a new patient with developmental delay and unusual clinical features.

De novo interstitial deletions of the short arm of chromosome 2 are rare chromosomal abnormalities. Patients showing these kind of microdeletions have developmental delay/intellectual disability, minor facial anomalies including high forehead, frontal bossing, broad nasal bridge, abnormal ears and congenital defects such as skeletal and genital malformations. We describe the second child of a healthy and non consanguineous couple presenting at birth multiple malformations and minor facial anomalies. Because of the clinical findings, an array CGH analysis was performed using Agilent 60K microarray oligonucleotide. The analysis detected a 9.3 Mb deletion on the short arm of chromosome 2 at band p11.2p12 spanning the bases 77,946,599-87,277,610. The five patients previously described display a minimal common deleted region which explains the clinical features shared by all of them, while their individual characteristics might be explained by the different sizes of the deletion. The common deleted region involves several genes (CTNNA2, LRRTM1, REEP1), highly expressed in the nervous system. The deletion found in this case overlaps with most of those reported in literature but our patient displays extra clinical signs such as bilateral choanal atresia and atrial septal defect. It was impossible to find any direct correlation between the genes involved in the deletion and the choanal atresia and the heart defect. The question remains open as to whether these clinical features are a consequence of the deletion or are due to a second pathogenic event. Our case emphasizes the difficulties to find a close correlation between a large deletion and a well defined clinical picture. As only five patients with 2p11.2p12 deletions, reported in the literature are characterized by array CGH, further reports will be necessary to well define a clinical phenotype related to the 2p11.2p12 microdeletion.

A comprehensive look at transcription factor gene expression changes in colorectal adenomas.

BACKGROUND: Biological processes are controlled by transcription networks. Expression changes of transcription factor (TF) genes in precancerous lesions are therefore crucial events in tumorigenesis. Our aim was to obtain a comprehensive picture of these changes in colorectal adenomas. METHODS: Using a 3-pronged selection procedure, we analyzed transcriptomic data on 34 human tissue samples (17 adenomas and paired samples of normal mucosa, all collected with ethics committee approval and written, informed patient consent) to identify TFs with highly significant tumor-associated gene expression changes whose potential roles in colorectal tumorigenesis have been under-researched. Microarray data were subjected to stringent statistical analysis of TF expression in tumor vs. normal tissues, MetaCore-mediated identification of TF networks displaying enrichment for genes that were differentially expressed in tumors, and a novel quantitative analysis of the publications examining the TF genes' roles in colorectal tumorigenesis. RESULTS: The 261 TF genes identified with this procedure included DACH1, which plays essential roles in the proper proliferation and differentiation of retinal and leg precursor cell populations in Drosophila melanogaster. Its possible roles in colorectal tumorigenesis are completely unknown, but it was found to be markedly overexpressed (mRNA and protein) in all colorectal adenomas and in most colorectal carcinomas. However, DACH1 expression was absent in some carcinomas, most of which were DNA mismatch-repair deficient. When networks were built using the set of TF genes identified by all three selection procedures, as well as the entire set of transcriptomic changes in adenomas, five hub genes (TGFB1, BIRC5, MYB, NR3C1, and TERT) where identified as putatively crucial components of the adenomatous transformation process. CONCLUSION: The transcription-regulating network of colorectal adenomas (compared with that of normal colorectal mucosa) is characterized by significantly altered expression of over 250 TF genes, many of which have never been investigated in relation to colorectal tumorigenesis.

Molecular pathways undergoing dramatic transcriptomic changes during tumor development in the human colon.

BACKGROUND: The malignant transformation of precancerous colorectal lesions involves progressive alterations at both the molecular and morphologic levels, the latter consisting of increases in size and in the degree of cellular atypia. Analyzing preinvasive tumors of different sizes can therefore shed light on the sequence of these alterations. METHODS: We used a molecular pathway-based approach to analyze transcriptomic profiles of 59 colorectal tumors representing early and late preinvasive stages and the invasive stage of tumorigenesis. Random set analysis was used to identify biological pathways enriched for genes differentially regulated in tumors (compared with 59 samples of normal mucosa). RESULTS: Of the 880 canonical pathways we investigated, 112 displayed significant tumor-related upregulation or downregulation at one or more stages of tumorigenesis. This allowed us to distinguish between pathways whose dysregulation is probably necessary throughout tumorigenesis and those whose involvement specifically drives progression from one stage to the next. We were also able to pinpoint specific changes within each gene set that seem to play key roles at each transition. The early preinvasive stage was characterized by cell-cycle checkpoint activation triggered by DNA replication stress and dramatic downregulation of basic transmembrane signaling processes that maintain epithelial/stromal homeostasis in the normal mucosa. In late preinvasive lesions, there was also downregulation of signal transduction pathways (e.g., those mediated by G proteins and nuclear hormone receptors) involved in cell differentiation and upregulation of pathways governing nuclear envelope dynamics and the G2>M transition in the cell cycle. The main features of the invasive stage were activation of the G1>S transition in the cell cycle, upregulated expression of tumor-promoting microenvironmental factors, and profound dysregulation of metabolic pathways (e.g., increased aerobic glycolysis, downregulation of pathways that metabolize drugs and xenobiotics). CONCLUSIONS: Our analysis revealed specific pathways whose dysregulation might play a role in each transition of the transformation process. This is the first study in which such an approach has been used to gain further insights into colorectal tumorigenesis. Therefore, these data provide a launchpad for further exploration of the molecular characterization of colorectal tumorigenesis using systems biology approaches.

The expression of leucine-rich repeat gene family members in colorectal cancer.

This study was conducted to evaluate the association of the leucine-rich repeat (LRR) gene family with colorectal cancer (CRC). The expression of members of the LRR gene family were analyzed in 17 CRC specimens and in 59 healthy colorectal tissues by using Human Exon1.0ST microarray, and in 25 CRC specimens and 32 healthy colorectal tissues by U133Plus2.0 microarray. An association was found for 25 genes belonging to the plant-specific (PS) class of LRR genes (P = 0.05 for Exon1.0 ST and P = 0.04 for U133Plus2.0). In both data-sets, in CRC, we found down-regulation of SHOC2 (P < 0.00003) and LRRC28 (P < 0.01) and up-regulation of LRSAM1 (P < 0.000001), while up-regulation of MFHAS1 (P = 0.0005) and down-regulation of WDFY3 (P = 0.026) were found only in the Exon1.0 ST data-set. The PS LLR gene class encodes proteins that activate immune cells and might play a key role in programmed cell death and autophagy. SHOC2 and LRRC28 genes involved in RAS-mediated signaling, which hinders nutrient deprivation-induced autophagy, might be a possible link between the negative control of autophagy and tumorigenesis.