A novel case of 16q22.3 duplication syndrome in a child with overgrowth: case report and literature review.

BACKGROUND: Distal chromosome 16 duplication syndrome (also known as 16q partial trisomy) is a very rare genetic disorder recently described in few clinical reports. 16q trisomy is generally associated with a multisystemic phenotype including intrauterine growth restriction (IUGR), brain and cardiac defects, intellectual disability (ID) and an increased risk of both prenatal and postnatal lethality. Smaller copy number variants (CNV) within the 16q region create partial trisomies, which occur less frequently than full trisomy 16q. CASE PRESENTATION: We present the clinical case of a 12-years-old male with a 16q22.3q24.1 de novo heterozygous duplication whose phenotype was characterized by ID, facial dysmorphisms, stature and weight overgrowth. To date, only five other cases of this syndrome have been reported in scientific literature, and none of them comprised overgrowth. CONCLUSIONS: Our case report highlights the great heterogeneity in clinical manifestations and provides new evidence for better defining the phenotypic picture for smaller 16q distal CNVs, suggesting unusual features.

Deep Intronic LINE-1 Insertions in NF1: Expanding the Spectrum of Neurofibromatosis Type 1-Associated Rearrangements.

Neurofibromatosis type 1 is an autosomal-dominant condition caused by NF1 gene inactivation. Clinical diagnosis is corroborated by genetic tests on gDNA and cDNA, which are inconclusive in approximately 3-5% of cases. Genomic DNA approaches may overlook splicing-affecting intronic variants and structural rearrangements, especially in regions enriched in repetitive sequences. On the other hand, while cDNA-based methods provide direct information about the effect of a variant on gene transcription, they are hampered by non-sense-mediated mRNA decay and skewed or monoallelic expression. Moreover, analyses on gene transcripts in some patients do not allow tracing back to the causative event, which is crucial for addressing genetic counselling, prenatal monitoring, and developing targeted therapies. We report on a familial NF1, caused by an insertion of a partial LINE-1 element inside intron 15, leading to exon 15 skipping. Only a few cases of LINE-1 insertion have been reported so far, hampering gDNA studies because of their size. Often, they result in exon skipping, and their recognition of cDNA may be difficult. A combined approach, based on Optical Genome Mapping, WGS, and cDNA studies, enabled us to detect the LINE-1 insertion and test its effects. Our results improve knowledge of the NF1 mutational spectrum and highlight the importance of custom-built approaches in undiagnosed patients.

A Complex Genomic Rearrangement Resulting in Loss of Function of SCN1A and SCN2A in a Patient with Severe Developmental and Epileptic Encephalopathy.

Complex genomic rearrangements (CGRs) are structural variants arising from two or more chromosomal breaks, which are challenging to characterize by conventional or molecular cytogenetic analysis (karyotype and FISH). The integrated approach of standard and genomic techniques, including optical genome mapping (OGM) and genome sequencing, is crucial for disclosing and characterizing cryptic chromosomal rearrangements at high resolutions. We report on a patient with a complex developmental and epileptic encephalopathy in which karyotype analysis showed a de novo balanced translocation involving the long arms of chromosomes 2 and 18. Microarray analysis detected a 194 Kb microdeletion at 2q24.3 involving the SCN2A gene, which was considered the likely translocation breakpoint on chromosome 2. However, OGM redefined the translocation breakpoints by disclosing a paracentric inversion at 2q24.3 disrupting SCN1A. This combined genomic high-resolution approach allowed a fine characterization of the CGR, which involves two different chromosomes with four breakpoints. The patient's phenotype resulted from the concomitant loss of function of SCN1A and SCN2A.

Identifying phenotypic expansions for congenital diaphragmatic hernia plus (CDH+) using DECIPHER data.

Congenital diaphragmatic hernia (CDH) can occur in isolation or in conjunction with other birth defects (CDH+). A molecular etiology can only be identified in a subset of CDH cases. This is due, in part, to an incomplete understanding of the genes that contribute to diaphragm development. Here, we used clinical and molecular data from 36 individuals with CDH+ who are cataloged in the DECIPHER database to identify genes that may play a role in diaphragm development and to discover new phenotypic expansions. Among this group, we identified individuals who carried putatively deleterious sequence or copy number variants affecting CREBBP, SMARCA4, UBA2, and USP9X. The role of these genes in diaphragm development was supported by their expression in the developing mouse diaphragm, their similarity to known CDH genes using data from a previously published and validated machine learning algorithm, and/or the presence of CDH in other individuals with their associated genetic disorders. Our results demonstrate how data from DECIPHER, and other public databases, can be used to identify new phenotypic expansions and suggest that CREBBP, SMARCA4, UBA2, and USP9X play a role in diaphragm development.

Reciprocal Xp11.4p11.3 microdeletion/microduplication spanning USP9X, DDX3X, and CASK genes in two patients with syndromic intellectual disability.

Only a few patients with deletions or duplications at Xp11.4, bridging USP9X, DDX3X, and CASK genes, have been described so far. Here, we report on a female harboring a de novo Xp11.4p11.3 deletion and a male with an overlapping duplication inherited from an unaffected mother, presenting with syndromic intellectual disability. We discuss the role of USP9X, DDX3X, and CASK genes in human development and describe the effects of Xp11.4 deletion and duplications in female and male patients, respectively.

8p23.1 deletion: Look out for left ventricular hypertrabeculation and not only congenital heart diseases. Single-center experience and literature revision.

Deletions involving the distal portion of the short arm of chromosome 8(8p23.1) show a high phenotypic variability. Congenital heart diseases (CHD) are often described. GATA4 when mutated or deleted is reported to be involved in cardiac morphogenesis. Only twice, left ventricular non compaction (LVNC) was reported in literature in association with 8p23.1 deletion. The present cohort includes five new patients with 8p23.1 deletions including GATA4. The spectrum of CHD is variable. Moreover, in four patients, LV hypertrabeculation was detected and in the fifth LVNC was recognized. Literature revision identified 45 patients with 8p23.1 deletions (encompassing GATA4) and heart involvement. It included wide spectrum of CHD including: heterotaxy spectrum 7/45 (15, 6%), atrioventricular canal 14/45 (balanced 3/45 including two of them with hypoplastic aortic arch; unbalanced 4/45, Fallot-AVC 1/45, partial AVC 3/45, unspecified 3/45), predominant major left heart lesions included 2/45 (4, 4%): interrupted aortic arch and hypoplastic left heart syndrome. Left ventricular hypertrabeculation might be potentially underestimated in patients with 8p23.1 deletion. These might suggest the importance of including microarray analysis in this group of patients. Moreover, 8p23.1 microdeletion or GATA4 variants can be considered in heterotaxy genetic panels.

1p36 Deletion Syndrome and the Aorta: A Report of Three New Patients and a Literature Review.

BACKGROUND: Monosomy 1p36 syndrome is now considered the most common terminal deletion syndrome, with an estimated incidence of 1 in 5000. Cardiac involvement is well described in the literature mainly in terms of congenital heart defects (CHDs) and cardiomyopathies (CMPs). Few data in the literature describe the potential progressive nature of aortic dilatation (root and ascending aorta) in 1p36 deletion syndrome. SKI harboured in the deleted region might play a predisposing factor for this aspect. METHODS: we reviewed the aortic aspect both in the literature and in our cohort, where major attention to the aortic abnormalities was given through dedicated echocardiographic measurements even in previously screened individuals. RESULTS: aortic involvement in 1p36 deletion syndrome was described in the literature three times within the CHD context. We observed three additional patients from our cohort (three out of nine patients) with aortic dilatation. All patients with dilated aorta had SKI haploinsufficiency within the deleted region. CONCLUSIONS: at long-term outcome and with a growing population of this rare disease, this association (1p36 deletion and aortic dilatation) might represent a major concern especially in terms of risk stratification and the potential need for specific management (conservative pharmacologic and eventually surgical) whenever indicated. The present study suggests the need for detailed multicentric studies and indication to periodic echocardiographic screening in addition to baseline tests, especially in individuals with deletions harbouring SKI.

Homozygous HESX1 and COL1A1 Gene Variants in a Boy with Growth Hormone Deficiency and Early Onset Osteoporosis.

We report on a patient born to consanguineous parents, presenting with Growth Hormone Deficiency (GHD) and osteoporosis. SNP-array analysis and exome sequencing disclosed long contiguous stretches of homozygosity and two distinct homozygous variants in HESX1 (Q6H) and COL1A1 (E1361K) genes. The HESX1 variant was described as causative in a few subjects with an incompletely penetrant dominant form of combined pituitary hormone deficiency (CPHD). The COL1A1 variant is rare, and so far it has never been found in a homozygous form. Segregation analysis showed that both variants were inherited from heterozygous unaffected parents. Present results further elucidate the inheritance pattern of HESX1 variants and recommend assessing the clinical impact of variants located in C-terminal propeptide of COL1A1 gene for their potential association with rare recessive and early onset forms of osteoporosis.

Clinical Application of Easychip 8x15K Platform in 4106 Pregnancies Without Ultrasound Anomalies.

Clinical utility of Array-CGH Easychip 8x15K platform can be assessed by testing its ability to detect the occurrence of pathogenic copy number variants (CNVs), and occurrence of variants of uncertain significance (VoUS) in pregnancies without structural fetal malformations. The demand of chromosomal microarray analysis in prenatal diagnosis is progressively increasing in uneventful pregnancies. However, depending on such platform resolution, a genome-wide approach also provides a high risk of detecting VoUS and incidental finding (IF) also defined as "toxic findings." In this context, novel alternative strategies in probe design and data filtering are required to balance the detection of disease causing CNVs and the occurrence of unwanted findings. In a cohort of consecutive pregnancies without ultrasound anomalies, a total of 4106 DNA samples from cultured and uncultured amniotic fluid or chorionic villi were collected and analyzed by a previously designed Array-CGH mixed-resolution custom platform, which is able to detect pathogenic CNVs and structural imbalanced rearrangements limiting the identification of VoUS and IF. Pathogenic CNVs were identified in 88 samples (2.1%), 19 of which (0.5%) were undetectable by standard karyotype. VoUS accounted for 0.6% of cases. Our data confirm that a mixed-resolution and targeted array CGH platform, as Easychip 8x15K, yields a similar detection rate of higher resolution CMA platforms and reduces the occurrence of "toxic findings," hence making it eligible for a first-tier genetic test in pregnancies without ultrasound anomalies.

Atypical 7q11.23 deletions excluding ELN gene result in Williams-Beuren syndrome craniofacial features and neurocognitive profile.

Williams-Beurens syndrome (WBS) is a rare genetic disorder caused by a recurrent 7q11.23 microdeletion. Clinical characteristics include typical facial dysmorphisms, weakness of connective tissue, short stature, mild to moderate intellectual disability and distinct behavioral phenotype. Cardiovascular diseases are common due to haploinsufficiency of ELN gene. A few cases of larger or smaller deletions have been reported spanning towards the centromeric or the telomeric regions, most of which included ELN gene. We report on three patients from two unrelated families, presenting with distinctive WBS features, harboring an atypical distal deletion excluding ELN gene. Our study supports a critical role of CLIP2, GTF2IRD1, and GTF2I gene in the WBS neurobehavioral profile and in craniofacial features, highlights a possible role of HIP1 in the autism spectrum disorder, and delineates a subgroup of WBS individuals with an atypical distal deletion not associated to an increased risk of cardiovascular defects.

PPP1R21-related syndromic intellectual disability: Report of an adult patient and review.

Variants in PPP1R21 were recently found to be associated with an autosomal recessive intellectual disability syndrome in 9 individuals. Our patient, the oldest among the known subjects affected by PPP1R21-related syndrome, manifested intellectual disability, short stature, congenital ataxia with cerebellar vermis hypoplasia, generalized hypertrichosis, ulcerative keratitis, muscle weakness, progressive coarse appearance, macroglossia with fissured tongue, and deep palmar and plantar creases. We provide an overview of the clinical spectrum and natural history of this newly recognized disorder, arguing the emerging notion that PPP1R21 gene mutations could result in endolysosomal functional defects. The oldest patients could display a more severe clinical outcome, due to accumulation of metabolites or damage secondary to an alteration of the autophagy pathway. Follow-up of patients with PPP1R21 mutations is recommended for improving the understanding of PPP1R21-related syndromic intellectual disability.

Hypermethioninemia in Campania: Results from 10 years of newborn screening.

In the last years tandem mass spectrometry (MS/MS) has become a leading technology used for neonatal screening purposes. Newborn screening by MS/MS on dried blood spot samples (DBS) has one of its items in methionine levels: the knowledge of this parameter allows the identification of infant affected by homocystinuria (cystathionine ß-synthase, CBS, deficiency) but can also lead, as side effect, to identify cases of methionine adenosyltransferase (MAT) type I/III deficiency. We started an expanded newborn screening for inborn errors of metabolism in Campania region in 2007. Here we report our ten years experience on expanded newborn screening in identifying patients affected by hypermethioninemia. During this period we screened approximately 77,000 infants and identified two cases: one case of classical homocystinuria and one patient affected by defect of MAT I/III. In this paper we describe these patients and their biochemical follow-up and review the literature concerning worldwide newborn screening reports on incidence of CBS and MAT deficiency.

DP71 and SERCA2 alteration in human neurons of a Duchenne muscular dystrophy patient.

Cognitive deficit has been identified in one third of patients affected by Duchenne Muscular Dystrophy, primarily attributed to loss of the short Dp71 dystrophin, the major brain dystrophin isoform. In this study, we investigated for the first time the Dp71 and Dp71-associated proteins cellular localization and expression in human neurons obtained by differentiation from induced pluripotent stem cell line of a patient affected by cognitive impairment. We found structural and molecular alterations in both pluripotent stem cell and derived neurons, reduced Dp71 expression, and a Ca2+ cytoplasmic overload in neurons coupled with increased expression of the SERCA2 pump in the dystrophic neurons. These results suggest that the reduction of Dp71 protein in the Duchenne muscular dystrophy neurons leads to alterations in SERCA2 and to elevated cytosolic Ca2+ concentration with consequent potential disruption of the dystrophin proteins and Dp71-associated proteins.

Gait disturbance and lower limb pain in a patient with PIK3CA-related disorder.

Post-zygotic activating mutations in PIK3CA and other genes encoding members of PI3K-AKT-mTOR pathway have been found in various overgrowth syndromes that have been grouped together as PIK3CA-related overgrowth spectrum (PROS). We report a female patient with gait disturbance, leg pain, isolated macrodactyly of the foot, and mild intellectual disability. Imaging of the lower limb showed a lipoblastoma of the right thigh. A mosaic gain-of-function mutation in the catalytic domain of PIK3CA (c.3140 A > G; p.His1047Arg) was detected in the adipose tissue and in skin cultured fibroblasts from the macrodactyly but not in blood. The leg pain and the severe walking disturbance improved slightly over time and serial MRI of the lower limbs suggested that the size of the lipoblastoma relative to the lower limb muscles or to the whole lower limb was unchanged as consequence of limb growth. This case report illustrates that pain and gait disturbance can be features of PROS and highlights the need of better knowledge about the natural history of the disease.

Angiogenesis and Vascularization of Uterine Leiomyoma: Clinical Value of Pseudocapsule Containing Peptides and Neurotransmitters.

Fibroids or myomas involve large proportion of women of reproductive age. The myoma formation starts from the transformation of the myometrium, causing the progressive formation of a pseudocapsule, which is made of compressed muscle fibers. Numerous studies investigated on myoma pseudocapsule anatomy, discovering many neurotransmitters and neuropeptides, as a neurovascular bundle, influencing myometrial physiology. These substances have a positive impact on wound healing and muscular restoring, also playing a role in sexual and reproductive function. Based on investigations, a distinct surgical technique evolved, called "intracapsular myomectomy", meaning myoma removal from its pseudocapsule, which enables protection of the myoma pseudocapsule, containing neuropeptides and neurofibers involved in physiological myometrial healing. This technique, performed by a gentle myoma enucleating by stretching from myometrium and sparing pseudocapsule, reduces surgical trauma caused by iatrogenic myoma pseudocapsule damage. Intracapsular myomectomy meets the basic surgical anatomy principle: myoma is removed by a bloodless, precise and careful dissection sparing myometrium, as much as possible. The rationale of intracapsular myomectomy should be applied to all myoma removals; therefore, it has been used for both laparoscopic and laparotomic myomectomy, as well as for cesarean myomectomy. Scientific research is still seeks to clarify some reports of myomas with infertility, especially in the case of intramural myomas, but it is clear that in the case of performing myomectomy, it must do by the described intracapsular technique. This enables myometrial preservation, especially peripherally to myoma bed, promoting myometrial healing after myoma removal.

A Proteomic Analysis of Human Uterine Myoma.

Uterine leiomyoma is a benign smooth muscle tumor characterized by a high incidence in women of reproductive age. The aetiology of this tumor is still unknown but established risk factors include high levels of female hormones, family history, African ancestry, early age of menarche and obesity. Here, to identify proteomic features associated with this tumor type, we performed a liquid chromatography-mass spectrometry (LC-MS/MS) analysis of uterine myomas. The identified proteins were subjected to a gene ontology analysis to generate biological functions, molecular processes, and protein networks that were relevant to the uploaded dataset. Pathway-based analysis was an effective approach to investigate the molecular mechanisms underlying the disease and to create biological hypotheses about regulation of our proteins including the identification of upstream regulators and main protein nodes. Moreover, proteomic and in silico data were combined with immunohistochemistry and western blotting to identify a group of proteins representative of some selected pathways, with a dysregulated expression in myoma, pseudocapsule, and normal myometrium samples. Based on these results, we confirmed the over-expression of extracellular matrix components, and estrogen and progesterone receptors in uterine myomas, and proposed biological networks, canonical pathways and functions that may be relevant to the pathophysiology of this tumor.

Laminin and Collagen IV: Two Polypeptides as Marker of Dystocic Labor.

Collagen IV and Laminin are localized in cells and tissue of numerous human organs including the uterus, where these polypeptides control either age changes, or uterus growth in pregnancy, or ripening and dilatation in labor. Authors examined the polypeptides distribution of collagen IV and Laminin in the human pregnant uterus, in normal and dystocic labor, to clarify their physiologic role, by distribution and/or their changes in prolonged dystocic labor. We collected lower uterine segment (LUS) fragments during cesarean section (CS); these biopsies were treated with basic morphological staining for the observation of microscopic- anatomic details. Other samples were processed with immunohistochemical staining for collagen IV and for membrane bound Laminin. All morphological and immunochemical results were analyzed with quantitative analysis of images and statistical analysis of data. Both Collagen IV and Laminin show changes in the pregnant uterus before 4 hours of full cervical dilatation in patients after 4 hours. All the three types of the human uterine cells, mucosal, submucosal and smooth muscular cells, are more reduced in LUS after 4 hours of cervical dilatation in dystocic labor. The connective tissues (including fibroblast) show the most evident changes in the dystocic LUS, collagen IV and laminin changes during cervical dilatation in prolonged dystocic labor, with a decreased elasticity with increased roughness and dryness. The LUS anatomical modifications during labor can be the cause of pathological changes in protracted dystocic labor. In the dystocic labor that lasts more than 4 hours from the complete cervical ripening and dilatation, the laminin and collagen IV concentration reduces in the LUS tissue. In dystocic labor, delivery should be completed before the 3 hours of full dilation, to avoid a reduction of laminin and collagen IV and a worsening of LUS healing for the next pregnancy.

Neurotransmitters and Neuropeptides Expression in the Uterine Scar After Cesarean Section.

Peptides and neuropeptides influence the uterine disorders of healing or cicatrization, chronic pelvic pain and disorder of pregnancy, labor and puerperium. They also promote changes in the lower uterine segment (LUS) during pregnancy, labor and delivery. We investigated the tissue quantity of neurotensin (NT), neuropeptide tyrosin (NPY) and Protein Gene Product 9.5 (PGP 9.5) in women submitted to elective cesarean section (CS) and urgent CS. During surgery, authors biopsied tissue samples of vesico-uterine space (VUS) to detect nerve fibers, and compared them. VUS samples from 106 patients have been evaluated with light microscopy, immunochemistry and Immunohistochemistry, and finally by Quantimet Leica analyzer software. Significantly higher amount of nerve fibers, containing NT, NPY and PGP 9.5 have been found in VUS tissue samples obtained during the first elective CS and during the first urgent CS were respectively 5±0.7, 7±0.6 and 5±0.9 CU and 2.5±0.5, 3.6±0.4 and 3.5±0.9 CU (p<0.05). This neurotransmitter reduction should indicate the inflammatory damage of cervical tissue for LUS over distension in dystocic-prolonged labor before CS. These results may be correlated with the decrease of NT, NPY and PGP 9.5, responsible for an optimal healing and LUS functions. In our opinion, the presence of neuropeptides reduction in uterine samples of women undergoing urgent CS may be due to a prolonged fetal head station in LUS, with a tissue denervation, in consequence of both overdistension and inflammatory process of the dystocic LUS.

A Qualitative and Quantitative Study of the Innervation of the Human Non Pregnant Uterus.

Human female reproductive system is closely dependent by hormonal stimulation. Anyway it is now commonly stated that autonomic innervation system regulates, along with hormonal stimulation, the uterine physiology. Cholinergic and adrenergic innervations have a critical role in mediating input to the uterus, but other neurotransmitters and neuropeptides exist that influence uterine physiology, as well. In the present investigation, we analyzed the uterine distribution of a large set of neurotransmitters, focusing on adrenergic, noradredenergic, acetylcholine (AChE) positive, dopaminergic, serotoninergic and peptidergic neurofibers; among these latter, we focused on those releasing prolattine, enkephalines (ENKs), Vasoactive Intestinal Polypeptide (VIP), substance P (SP) and oxytocine. Authors demonstrate the differential localization of these neurofibers in non pregnant uterine fundus, corpus and cervix, sampling myometrial assays of 31 patients submitted to hysterectomy. In fundus uteri, we observed a prevalence of prolactinergic (32.1 ± 1.4 Conventional Unit, C.U.) and adrenergic (36.4 ± 4.5 C.U.) neurofibers; in uterine body VIP positive neurofibers (32.6 ± 4.8 C.U.) and prolactinergic neurofibers (30.3 ± 1.2 C.U.) were the most represented. In uterine cervix, we detected the highest concentration of all the neurofibers analysed, with enkephalinergic neurofibers (94 ± 1.7 C.U.), oxitocinergic neurofibers (72.1 ± 5.1 C.U.), SP positive neurofibers (66.1 ± 4.4 C.U.), acetylcholine positive neurofibers (64.5± 3.6 C.U.), serotoninergic neurofibers (56.4 ± 3.9 C.U.) and VIP positive neurofibers (58.3 ± 5.2 C.U.) being the most expressed. This study demonstrates that uterine cervix harbors a higher concentration of almost all neurotransmitters, compared to the other two uterine anatomic sites. The uterine cervix is largely involved during pregnancy and labor, and the rich neurotransmitters density could contribute to confer to the cervix a proper potential plasticity, necessary for pregnancy and labour.

The Drosophila fragile X mental retardation protein participates in the piRNA pathway.

RNA metabolism controls multiple biological processes, and a specific class of small RNAs, called piRNAs, act as genome guardians by silencing the expression of transposons and repetitive sequences in the gonads. Defects in the piRNA pathway affect genome integrity and fertility. The possible implications in physiopathological mechanisms of human diseases have made the piRNA pathway the object of intense investigation, and recent work suggests that there is a role for this pathway in somatic processes including synaptic plasticity. The RNA-binding fragile X mental retardation protein (FMRP, also known as FMR1) controls translation and its loss triggers the most frequent syndromic form of mental retardation as well as gonadal defects in humans. Here, we demonstrate for the first time that germline, as well as somatic expression, of Drosophila Fmr1 (denoted dFmr1), the Drosophila ortholog of FMRP, are necessary in a pathway mediated by piRNAs. Moreover, dFmr1 interacts genetically and biochemically with Aubergine, an Argonaute protein and a key player in this pathway. Our data provide novel perspectives for understanding the phenotypes observed in Fragile X patients and support the view that piRNAs might be at work in the nervous system.