Low-pass whole genome sequencing as a cost-effective alternative to chromosomal microarray analysis for low- and middle-income countries.

Low-pass whole genome sequencing (LP-WGS) has been applied as alternative method to detect copy number variants (CNVs) in the clinical setting. Compared with chromosomal microarray analysis (CMA), the sequencing-based approach provides a similar resolution of CNV detection at a lower cost. In this study, we assessed the efficiency and reliability of LP-WGS as a more affordable alternative to CMA. A total of 1363 patients with unexplained neurodevelopmental delay/intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies were enrolled. Those patients were referred from 15 nonprofit organizations and university centers located in different states in Brazil. The analysis of LP-WGS at 1x coverage (>50kb) revealed a positive testing result in 22% of the cases (304/1363), in which 219 and 85 correspond to pathogenic/likely pathogenic (P/LP) CNVs and variants of uncertain significance (VUS), respectively. The 16% (219/1363) diagnostic yield observed in our cohort is comparable to the 15%-20% reported for CMA in the literature. The use of commercial software, as demonstrated in this study, simplifies the implementation of the test in clinical settings. Particularly for countries like Brazil, where the cost of CMA presents a substantial barrier to most of the population, LP-WGS emerges as a cost-effective alternative for investigating copy number changes in cytogenetics.

ZDHHC9 X-linked intellectual disability: Clinical and molecular characterization.

The ZDHHC9 gene encodes the Zinc Finger DHHC-Type Containing 9 protein that functions as a palmitoyltransferase. Variants in this gene have been reported as the cause of Raymond-type X-linked intellectual disability with only 16 families described in the literature. This study reviews molecular and clinical data from previously reported patients and reports the case of a 13-year-old patient with a splicing variant in ZDHHC9 presenting intellectual disability, developmental delay, facial dysmorphisms, and skeletal defects. Although intellectual disability and developmental delay with severe speech delay have been reported in all cases with available clinical data, the remaining clinical signs differ significantly between patients. Missense, nonsense, frameshift, and splicing variants, in addition to large exonic deletions, have been described suggesting a loss of function mechanism. Though variants are distributed in almost all exons, most missense and nonsense variants affect arginine residues located in the cytoplasmic domains of this transmembrane protein, suggesting possible mutational hotspots.

Phenotypic and mutational spectrum of ROR2-related Robinow syndrome.

Robinow syndrome is characterized by a triad of craniofacial dysmorphisms, disproportionate-limb short stature, and genital hypoplasia. A significant degree of phenotypic variability seems to correlate with different genes/loci. Disturbances of the noncanonical WNT-pathway have been identified as the main cause of the syndrome. Biallelic variants in ROR2 cause an autosomal recessive form of the syndrome with distinctive skeletal findings. Twenty-two patients with a clinical diagnosis of autosomal recessive Robinow syndrome were screened for variants in ROR2 using multiple molecular approaches. We identified 25 putatively pathogenic ROR2 variants, 16 novel, including single nucleotide variants and exonic deletions. Detailed phenotypic analyses revealed that all subjects presented with a prominent forehead, hypertelorism, short nose, abnormality of the nasal tip, brachydactyly, mesomelic limb shortening, short stature, and genital hypoplasia in male patients. A total of 19 clinical features were present in more than 75% of the subjects, thus pointing to an overall uniformity of the phenotype. Disease-causing variants in ROR2, contribute to a clinically recognizable autosomal recessive trait phenotype with multiple skeletal defects. A comprehensive quantitative clinical evaluation of this cohort delineated the phenotypic spectrum of ROR2-related Robinow syndrome. The identification of exonic deletion variant alleles further supports the contention of a loss-of-function mechanism in the etiology of the syndrome.

SHORT syndrome in an adult Brazilian patient.

We report an individual from Brazil with SHORT syndrome. The term SHORT stands for its common characteristics: short stature (S), hyperextensibility of joints, and/or inguinal hernia (H), ocular depression (O), Rieger anomaly (R), and teething delay (T). In addition to most of the clinical signs previously described in SHORT syndrome, the patient presented here also shows microcephaly and intellectual disability. Diagnosis was confirmed by exome sequencing revealing a novel heterozygous variant c.1456G>A (p.Ala486Thr) at PIK3R1. Human recombinant growth hormone (r-hGH) therapy was administered prior to diagnosis; however, the use of r-hGH may have had a role in anticipating and worsening the glucose metabolic profile in the patient, as previously described. This article contributes to providing a better understanding of the SHORT syndrome genotype and its correlation with the phenotype, by comparing with it other reported cases.

WNT Signaling Perturbations Underlie the Genetic Heterogeneity of Robinow Syndrome.

Locus heterogeneity characterizes a variety of skeletal dysplasias often due to interacting or overlapping signaling pathways. Robinow syndrome is a skeletal disorder historically refractory to molecular diagnosis, potentially stemming from substantial genetic heterogeneity. All current known pathogenic variants reside in genes within the noncanonical Wnt signaling pathway including ROR2, WNT5A, and more recently, DVL1 and DVL3. However, ∼70% of autosomal-dominant Robinow syndrome cases remain molecularly unsolved. To investigate this missing heritability, we recruited 21 families with at least one family member clinically diagnosed with Robinow or Robinow-like phenotypes and performed genetic and genomic studies. In total, four families with variants in FZD2 were identified as well as three individuals from two families with biallelic variants in NXN that co-segregate with the phenotype. Importantly, both FZD2 and NXN are relevant protein partners in the WNT5A interactome, supporting their role in skeletal development. In addition to confirming that clustered -1 frameshifting variants in DVL1 and DVL3 are the main contributors to dominant Robinow syndrome, we also found likely pathogenic variants in candidate genes GPC4 and RAC3, both linked to the Wnt signaling pathway. These data support an initial hypothesis that Robinow syndrome results from perturbation of the Wnt/PCP pathway, suggest specific relevant domains of the proteins involved, and reveal key contributors in this signaling cascade during human embryonic development. Contrary to the view that non-allelic genetic heterogeneity hampers gene discovery, this study demonstrates the utility of rare disease genomic studies to parse gene function in human developmental pathways.

Neurocognitive, adaptive, and psychosocial functioning in individuals with Robinow syndrome.

It has been estimated that 10-15% of people with Robinow syndrome (RS) show delayed development, but no studies have formally assessed developmental domains. The objective of this study is to provide the first description of cognitive, adaptive, and psychological functioning in RS. Thirteen participants (10 males) aged 4-51 years were seen for neuropsychological screening. Eight had autosomal-dominant RS (DVL1, n = 5; WNT5A, n = 3), four had autosomal-recessive RS (NXN, n = 2; ROR2, n = 2), and one had a mutation on an RS candidate gene (GPC4). Participants completed measures of intellectual, fine-motor, adaptive, executive, and psychological functioning. Findings indicated generally average intellectual functioning and low-average visuomotor skills. Adaptive functioning was average in autosomal-recessive RS (RRS) but low average in autosomal-dominant RS (DRS). Parent-report indicated executive dysfunction and attention problems in 4/8 children, 3/4 of whom had a DVL1 variant; adult self-report did not indicate similar difficulties. Learning disabilities were also reported in 4/8 individuals with DRS, 3/4 of whom had a DVL1 variant. Peer problems were reported for a majority of participants, many of whom also reported emotional concerns. Altogether, the findings indicate average neurocognitive functioning in RRS. In contrast, DRS, especially DVL1 pathogenic alleles, may confer specific risk for neurodevelopmental disability.

Novel pathogenic genomic variants leading to autosomal dominant and recessive Robinow syndrome.

Robinow syndrome (RS) is a genetically heterogeneous disorder characterized by skeletal dysplasia and a distinctive facial appearance. Previous studies have revealed locus heterogeneity with rare variants in DVL1, DVL3, FZD2, NXN, ROR2, and WNT5A underlying the etiology of RS. The aforementioned "Robinow-associated genes" and their gene products all play a role in the WNT/planar cell polarity signaling pathway. We performed gene-targeted Sanger sequencing, exome sequencing, genome sequencing, and array comparative genomic hybridization on four subjects with a clinical diagnosis of RS who had not had prior DNA testing. Individuals in our cohort were found to carry pathogenic or likely pathogenic variants in three RS related genes: DVL1, ROR2, and NXN. One subject was found to have a nonsense variant (c.817C > T [p.Gln273*]) in NXN in trans with an ~1 Mb telomeric deletion on chromosome 17p containing NXN, which supports our contention that biallelic NXN variant alleles are responsible for a novel autosomal recessive RS locus. These findings provide increased understanding of the role of WNT signaling in skeletal development and maintenance. These data further support the hypothesis that dysregulation of the noncanonical WNT pathway in humans gives rise to RS.

Characterization of the Robinow syndrome skeletal phenotype, bone micro-architecture, and genotype-phenotype correlations with the osteosclerotic form.

Robinow syndrome (RS) is a genetically heterogeneous skeletal dysplasia with recent reports suggesting an osteosclerotic form of the disease. We endeavored to investigate the full spectrum of skeletal anomalies in a genetically diverse cohort of RS patients with a focus on the bone micro-architecture. Seven individuals with molecularly confirmed RS, including four with DVL1 variants and single individuals with variants in WNT5A, ROR2, and GPC4 underwent a musculoskeletal focused physical examination, dual-energy X-ray absorptiometry (DEXA) scan, and high-resolution peripheral quantitative computed tomography (HR-pQCT). Skeletal examination revealed variability in limb shortening anomalies consistent with recent reports. DEXA scan measures revealed increased total body bone mineral density (BMD) (3/7), cranial BMD (5/7), and non-cranial BMD (1/7). Cranial osteosclerosis was only observed in DVL1-RS (4/4) and GPC4-RS (1/1) subjects and in one case was complicated by choanal atresia, bilateral conductive hearing loss, and cranial nerve III, VI, and VII palsy. HR-pQCT revealed a unique pattern of low cortical BMD, increased trabecular BMD, decreased number of trabeculations, and increased thickness of the trabeculations for the DVL1-RS subjects. The spectrum of skeletal anomalies including the micro-architecture of the bones observed in RS has considerable variability with some osteosclerosis genotype-phenotype correlations more frequent with DVL1 variants.

DVL3 Alleles Resulting in a -1 Frameshift of the Last Exon Mediate Autosomal-Dominant Robinow Syndrome.

Robinow syndrome is a rare congenital disorder characterized by mesomelic limb shortening, genital hypoplasia, and distinctive facial features. Recent reports have identified, in individuals with dominant Robinow syndrome, a specific type of variant characterized by being uniformly located in the penultimate exon of DVL1 and resulting in a -1 frameshift allele with a premature termination codon that escapes nonsense-mediated decay. Here, we studied a cohort of individuals who had been clinically diagnosed with Robinow syndrome but who had not received a molecular diagnosis from variant studies of DVL1, WNT5A, and ROR2. Because of the uniform location of frameshift variants in DVL1-mediated Robinow syndrome and the functional redundancy of DVL1, DVL2, and DVL3, we elected to pursue direct Sanger sequencing of the penultimate exon of DVL1 and its paralogs DVL2 and DVL3 to search for potential disease-associated variants. Remarkably, targeted sequencing identified five unrelated individuals harboring heterozygous, de novo frameshift variants in DVL3, including two splice acceptor mutations and three 1 bp deletions. Similar to the variants observed in DVL1-mediated Robinow syndrome, all variants in DVL3 result in a -1 frameshift, indicating that these highly specific alterations might be a common cause of dominant Robinow syndrome. Here, we review the current knowledge of these peculiar variant alleles in DVL1- and DVL3-mediated Robinow syndrome and further elucidate the phenotypic features present in subjects with DVL1 and DVL3 frameshift mutations.

Holoprosencephaly, orofacial cleft, and frontonaso-orbital encephaloceles: Genetic evaluation of a possible new syndrome.

Here we report on a Brazilian child who presented semilobar holoprosencephaly, frontonasal encephaloceles and bilateral cleft lip and palate. Malformations also included agenesis of the corpus callosum, abnormal cortical gyres, dilation of the aqueduct, bilateral endolymphatic sac, bilateral cystic cocci-vestibular malformation, and a cribriform defect. The 3D TC craniofacial images showed abnormal frontonasal transition region, with a bone bifurcation, and partial agenesis of nasal bone. The trunk and upper and lower limbs were normal. To our knowledge, this rare association of holoprocensephaly with frontonaso-orbital encephaloceles without limb anomalies has never been reported before. Karyotype was normal. SNP-array showed no copy-number alterations but revealed 25% of regions of homozygosity (ROH) with normal copy number, indicating a high coefficient of inbreeding, which significantly increases the risk for an autosomal recessive disorder. Whole exome sequencing analysis did not reveal any pathogenic or likely pathogenic variants. We discuss the possible influence of two variants of uncertain significance found within the patient's ROHs. First, a missense p.(Gly394Ser) in PCSK9, a gene involved in the regulation of plasma low-density lipoprotein cholesterol. Second, an inframe duplication p.(Ala75_Ala81dup) in SP8, a zinc-finger transcription factor that regulates signaling centers during craniofacial development. Further studies and/or the identification of other patients with a similar phenotype will help elucidate the genetic etiology of this complex case.

DVL1 frameshift mutations clustering in the penultimate exon cause autosomal-dominant Robinow syndrome.

Robinow syndrome is a genetically heterogeneous disorder characterized by mesomelic limb shortening, genital hypoplasia, and distinctive facial features and for which both autosomal-recessive and autosomal-dominant inheritance patterns have been described. Causative variants in the non-canonical signaling gene WNT5A underlie a subset of autosomal-dominant Robinow syndrome (DRS) cases, but most individuals with DRS remain without a molecular diagnosis. We performed whole-exome sequencing in four unrelated DRS-affected individuals without coding mutations in WNT5A and found heterozygous DVL1 exon 14 mutations in three of them. Targeted Sanger sequencing in additional subjects with DRS uncovered DVL1 exon 14 mutations in five individuals, including a pair of monozygotic twins. In total, six distinct frameshift mutations were found in eight subjects, and all were heterozygous truncating variants within the penultimate exon of DVL1. In five families in which samples from unaffected parents were available, the variants were demonstrated to represent de novo mutations. All variant alleles are predicted to result in a premature termination codon within the last exon, escape nonsense-mediated decay (NMD), and most likely generate a C-terminally truncated protein with a distinct -1 reading-frame terminus. Study of the transcripts extracted from affected subjects' leukocytes confirmed expression of both wild-type and variant alleles, supporting the hypothesis that mutant mRNA escapes NMD. Genomic variants identified in our study suggest that truncation of the C-terminal domain of DVL1, a protein hypothesized to have a downstream role in the Wnt-5a non-canonical pathway, is a common cause of DRS.

Short stature, unusual face, delta phalanx, and abnormal vertebrae and ribs in a girl born to half-siblings.

Delta phalanx is a rare abnormality typically associated with additional features. We describe a patient with a phenotype resembling Catel-Manzke syndrome, but with delta phalanx and abnormal vertebrae and ribs. The patient was the only child of half siblings born with a marked prenatal growth deficiency. At 10 years of age, she had a short stature, long face, long and tubular nose with small alae nasi, high palate, short and broad thorax, and short index fingers with radial deviation. There were hyperpigmentations following Blaschko's lines. Radiology showed a proximal delta phalanx in the index finger of hands, abnormal vertebrae, and fused and small ribs. GTG-Banding karyotype and microarray analysis yielded normal results. Exome sequencing identified 25 genes that harbored homozygous variants, but none of these is assumed to be a good candidate to explain (part of) the phenotype. The here described patient may have a new condition, possibly following an autosomal recessive pattern of inheritance, although due to the high degree of consanguinity a compound etiology of the phenotype by variants in various genes may be present as well.

Concurrent Loss of Heterozygosity and Mosaic Deletion of 12p13.32pter.

Deletions in the short arm of chromosome 12 are the rarest subtelomeric imbalances. Less than 20 patients have been reported to date, and their microdeletions were identified either by FISH or array-CGH without SNP data. Here, we report a patient with a 12p13.32pter mosaic deletion detected by chromosome microarray analysis with loss of heterozygosity (LOH) of the deleted segment in addition to the adjacent distal segment. LOH is indicative of a complex rearrangement, suggestive of mitotic microhomology-mediated break-induced replication.

Differential diagnosis between multiple sclerosis and leukodystrophies - A scoping review.

Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) characterized by damage to the myelin sheaths of oligodendrocytes. Currently, there is no specific biomarker to identify the disease; however, a diagnostic criterion has been established based on patient's clinical, laboratory, and imaging characteristics, which assists in identifying this condition. The primary method for diagnosing MS is the McDonald criteria, first described in 2001 and revised in the years 2005, 2012, and 2017. These criteria have been continuously reviewed to enhance specificity and sensitivity in the diagnosis of MS, thereby reducing errors in its differential diagnosis. An important differential diagnosis that shares overlapping features with MS, mainly the progressive forms, are leukodystrophies with demyelination as underlying pathology. Leukodystrophies comprise a rare group of genetically determined disorders that lead to either demyelination or hypomyelination of the central nervous system that can result neuroimaging changes as well as clinical findings similar to those observed in MS. Thus, systematic evaluation encompassing clinical presentation, neuroimaging findings, and laboratory metrics proves indispensable for a differential diagnosis. As such, this study aimed to establish, clearly and objectively, the similarities and differences between MS and the main demyelinating leukodystrophies. The study analyzed the parameters of the McDonald criteria, including clinical, laboratory, and magnetic resonance imaging aspects, as found in patients with leukodystrophies through scoping literature review. The data were compared with the determinations of the revised 2017 McDonald criteria to facilitate the differential diagnosis of these diseases in clinical practice.

Single nucleotide polymorphisms to predict acute radiation dermatitis in breast cancer patients: A systematic review and meta-analysis.

PURPOSE: To identify Single Nucleotide Polymorphisms (SNPs) that can predict acute radiation dermatitis (RD) in breast cancer patients (BC), and the association between SNPs and RD severity. METHODS: We performed the search in seven databases and the gray literature, and a meta-analysis to assess SNPs in patients with RD and to evaluate the association between SNPs and severe RD. RESULTS: We included sixteen single-arm cohort studies with 4742 BC. The most prevalent SNPs were the TGFß1 rs1800469 (41%), and the GSTA1 rs3957356 (36%). Seven genotypes were associated with severe RD (PTTG1 rs3811999-CC; PTTG1 rs2961950-AA; MAD2L2 rs2294638-GG; MAT1A rs2282367-GG; GSTA1 rs3957356-CT; CD44 rs8193-CT; SH3GL1 rs243336-GC) and five SNPs were associated with lower RD (PTTG1 rs2961952-GG; CD44 rs8193-CC; PTTG1 rs3811999-CT; MAT1A rs2282367-GA; OGG1 rs2075747-AA). CONCLUSIONS: The genotyping of SNPs more prevalent may be a strategy for predicting RD in BC, and some genotypes (GSTA1 rs3957356-CT; MAT1A rs2282367-GG) are associated with severe RD.

Characterization of Associated Nonclassical Phenotypes in Patients with Deletion in the WAGR Region Identified by Chromosomal Microarray: New Insights and Literature Review.

WAGR syndrome (Wilms' tumor, aniridia, genitourinary changes, and intellectual disability) is a contiguous gene deletion syndrome characterized by the joint deletion of PAX6 and WT1 genes, located in the short arm of chromosome 11. However, most deletions include other genes, leading to multiple associated phenotypes. Therefore, understanding how genes deleted together can contribute to other clinical phenotypes is still considered a challenge. In order to establish genotype-phenotype correlation in patients with interstitial deletions of the short arm of chromosome 11, we selected 17 patients with deletions identified by chromosomal microarray analysis: 4 new subjects and 13 subjects previously described in the literature with detailed clinical data. Through the analysis of deleted regions and the phenotypic changes, it was possible to suggest the contribution of specific genes to several nonclassical phenotypes, contributing to the accuracy of clinical characterization of the syndrome and emphasizing the broad phenotypic spectrum found in the patients. This study reports the first patient with a PAX6 partial deletion who does not present any eye anomaly thus opening a new set of questions about the functional activity of PAX6.

Novel pathogenic variants and quantitative phenotypic analyses of Robinow syndrome: WNT signaling perturbation and phenotypic variability.

Robinow syndrome (RS) is a genetically heterogeneous disorder with six genes that converge on the WNT/planar cell polarity (PCP) signaling pathway implicated (DVL1, DVL3, FZD2, NXN, ROR2, and WNT5A). RS is characterized by skeletal dysplasia and distinctive facial and physical characteristics. To further explore the genetic heterogeneity, paralog contribution, and phenotypic variability of RS, we investigated a cohort of 22 individuals clinically diagnosed with RS from 18 unrelated families. Pathogenic or likely pathogenic variants in genes associated with RS or RS phenocopies were identified in all 22 individuals, including the first variant to be reported in DVL2. We retrospectively collected medical records of 16 individuals from this cohort and extracted clinical descriptions from 52 previously published cases. We performed Human Phenotype Ontology (HPO) based quantitative phenotypic analyses to dissect allele-specific phenotypic differences. Individuals with FZD2 variants clustered into two groups with demonstrable phenotypic differences between those with missense and truncating alleles. Probands with biallelic NXN variants clustered together with the majority of probands carrying DVL1, DVL2, and DVL3 variants, demonstrating no phenotypic distinction between the NXN-autosomal recessive and dominant forms of RS. While phenotypically similar diseases on the RS differential matched through HPO analysis, clustering using phenotype similarity score placed RS-associated phenotypes in a unique cluster containing WNT5A, FZD2, and ROR2 apart from non-RS-associated paralogs. Through human phenotype analyses of this RS cohort and OMIM clinical synopses of Mendelian disease, this study begins to tease apart specific biologic roles for non-canonical WNT-pathway proteins.

Microduplication of the ICR2 domain at chromosome 11p15 and familial Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is characterized by severe intrauterine and postnatal growth retardation in association with a typical small triangular face and other variable features. Genetic and epigenetic disturbances are detected in about 50% of the patients. Most frequently, SRS is caused by altered gene expression on chromosome 11p15 due to hypomethylation of the telomeric imprinting center (ICR1) that is present in at least 40% of the patients. Maternally inherited duplications encompassing ICR1 and ICR2 domains at 11p15 were found in a few patients, and a microduplication restricted to ICR2 was described in a single SRS child. We report on a microduplication of the ICR2 domain encompassing the KCNQ1, KCNQ1OT1, and CDKN1C genes in a three-generation family: there were four instances of paternal transmissions of the microduplication from a single male uniformly resulting in normal offspring, and five maternal transmissions, via two clinically normal sisters, with all the children exhibiting SRS. This report provides confirmatory evidence that a microduplication restricted to the ICR2 domain results in SRS when maternally transmitted.