Exome sequencing in the pediatric neuromuscular clinic leads to more frequent diagnosis of both neuromuscular and neurodevelopmental conditions.

INTRODUCTION/AIMS: Exome sequencing (ES) has proven to be a valuable diagnostic tool for neuromuscular disorders, which often pose a diagnostic challenge. The aims of this study were to investigate the clinical outcomes associated with utilization of ES in the pediatric neuromuscular clinic and to determine if specific phenotypic features or abnormal neurodiagnostic tests were predictive of a diagnostic result. METHODS: This was a retrospective medical record review of 76 pediatric neuromuscular clinic patients who underwent ES. Based upon clinical assessment prior to ES, patients were divided into two groups: affected by neuromuscular (n = 53) or non-neuromuscular (n = 23) syndromes. RESULTS: A diagnosis was made in 28/76 (36.8%), with 29 unique disorders identified. In the neuromuscular group, a neuromuscular condition was confirmed in 78% of those receiving a genetic diagnosis. Early age of symptom onset was associated with a significantly higher diagnostic yield. The most common reason neuromuscular diagnoses were not detected on prior testing was due to causative genes not being present on disease-specific panels. Changes to medical care were made in 57% of individuals receiving a diagnosis on ES. DISCUSSION: These data further support ES as a powerful diagnostic tool in the pediatric neuromuscular clinic and highlight the advantages of ES over gene panels, including the ability to identify diagnoses regardless of etiology, identify genes newly associated with disease, and identify multiple confounding diagnoses. Rapid and accurate diagnosis by ES can not only end the patient's diagnostic odyssey, but often impacts patients' medical management and genetic counseling of families.

Idiopathic intracranial hypertension presenting as postpartum headache.

Postpartum headache is described as headache and neck or shoulder pain during the first 6 weeks after delivery. Common causes of headache in the puerperium are migraine headache and tension headache; other causes include pre-eclampsia/eclampsia, post-dural puncture headache, cortical vein thrombosis, subarachnoid hemorrhage, posterior reversible leukoencephalopathy syndrome, brain tumor, cerebral ischemia, meningitis, and so forth. Idiopathic intracranial hypertension (IIH) is a rare cause of postpartum headache. It is usually associated with papilledema, headache, and elevated intracranial pressure without any focal neurologic abnormality in an otherwise healthy person. It is more commonly seen in obese women of reproductive age group, but rare during pregnancy and postpartum. We present a case of IIH who presented to us 18 days after cesarean section with severe headache and was successfully managed.

Is there a difference in the clinical profile and outcome of women using levonorgestrel IUD for abnormal uterine bleeding and those using it for contraception?: A comparative cross-sectional study.

Objective: The most common indications for Levonorgestrel intrauterine device (LNG-IUD) are contraception and management of abnormal uterine bleeding (AUB). This study was conducted with the aim of exploring the differences in the clinical profile and outcome of women using LNG-IUD for contraception and AUB. Materials and Methods: This was a retrospective comparative cross-sectional study of women who underwent LNG-IUD (52 mg) between 2012 and 2017. Their electronic health records were reviewed until the last documented follow-up or until December 2021. Results: A total of 235 women had LNG-IUD with an age range of 21 to 62 years and a mean of (37.98 years±6.76). Of these women, 153/235 (65.1%) had it for contraception and 82/235 (34.89%) had it for AUB. The follow-up was 1-94 months with (mean ± SEM) follow-up for the AUB group of (21.48±2.31) months and for contraception group was (20.74±1.76) months (p-value of 0.80). There was a significant difference between the two groups in the age and body mass index (BMI), where women who had LNG-IUD for AUB were older (mean of 42.54±6.49 years, p-value <0.001) and had higher BMI (31.88±7.52 kg/m2, p-value =0.011). All LNG-IUDs that were indicated for contraception were inserted in an outpatient setting. However, 68.3% in the AUB, the insertion was in the operating theater in conjunction with hysteroscopy. After combining both expulsion and removal of LNG-IUD during the follow-up period, there was no significant difference between the 2 groups in the overall retention rate during the follow-up (p-value =0.998). Conclusion: this study shows that women using LNG-IUD for the management of AUB are older and have a higher BMI compared with those using it for contraception. AUB women experienced more expulsion compared with the contraception group, but there was no difference between the 2 groups in the overall survival/retention of LNG-IUD.

Clinical Cytogenetics: Current Practices and Beyond.

BACKGROUND: Throughout history, the field of cytogenetics has witnessed significant changes due to the constant evolution of technologies used to assess chromosome number and structure. Similar to the evolution of single nucleotide variant detection from Sanger sequencing to next-generation sequencing, the identification of chromosome alterations has progressed from banding to fluorescence in situ hybridization (FISH) to chromosomal microarrays. More recently, emerging technologies such as optical genome mapping and genome sequencing have made noteworthy contributions to clinical laboratory testing in the field of cytogenetics. CONTENT: In this review, we journey through some of the most pivotal discoveries that have shaped the development of clinical cytogenetics testing. We also explore the current test offerings, their uses and limitations, and future directions in technology advancements. SUMMARY: Cytogenetics methods, including banding and targeted assessments like FISH, continue to hold crucial roles in cytogenetic testing. These methods offer a rapid turnaround time, especially for conditions with a known etiology involving recognized cytogenetic aberrations. Additionally, laboratories have the flexibility to now employ higher-throughput methodologies to enhance resolution for cases with greater complexity.

Papillary Hemangioma Harbors Somatic GNA11 and GNAQ Mutations.

Papillary hemangioma (PH) is a small, primarily dermal lesion occurring predominantly in the head and neck in both children and adults. Its signature characteristics are dilated thin-walled channels containing papillary clusters of mainly capillary-sized vessels and endothelial cytoplasmic eosinophilic inclusions. Given certain histopathologic similarities to congenital hemangioma which harbor mutations in GNAQ and GNA11 , we investigated whether similar mutations are present in PH. Seven PH specimens were studied. All presented in the first 4 years of life, with one being noted at birth. With the exception of one lesion, all were in the head and neck. Lesions were bluish and ranged in size from 0.5 to 2.8 cm. Four samples had GNA11 p.Q209L and 3 had GNAQ p.Q209L missense mutations. Mutations in GNA11 and GNAQ are associated with other types of somatic vascular lesions including capillary malformation, congenital hemangioma, anastomosing hemangioma, thrombotic anastomosing hemangioma, and hepatic small cell neoplasm. Shared mutations in GNA11 and GNAQ may account for some overlapping clinical and pathologic features in these entities, perhaps explicable by the timing of the mutation or influence of the germline phenotype.

Multisite Verification of a Targeted CFTR Polymerase Chain Reaction/Capillary Electrophoresis Assay That Evaluates Pathogenic Variants Across Diverse Ethnic and Ancestral Groups.

CONTEXT.­: Existing targeted cystic fibrosis screening assays miss important pathogenic CFTR variants in the ethnically diverse US population. OBJECTIVE.­: To evaluate the analytic performance of a multiplex polymerase chain reaction (PCR)/capillary electrophoresis (CE) CFTR assay panel that simultaneously interrogates primary pathogenic variants of different ethnic/ancestral groups. DESIGN.­: Performance characteristic assessment and variant coverage comparison of the panel with a focus on ethnicity-specific CFTR variants were performed. Sample DNA was primarily from whole blood or cell lines. Detection of CFTR carriers was compared across several commercially available CFTR kits and recommended variant sets based on panel content. RESULTS.­: The panel interrogated 65 pathogenic CFTR variants representing 92% coverage from a recent genomic sequencing survey of the US population, including 4 variants with top 5 frequency in African or Asian populations not reflected in other targeted panels. In simulation studies, the panel represented 95% of carriers across the global population, resulting in 6.9% to 19.0% higher carrier detection rate compared with 10 targeted panels or variant sets. Precision and sensitivity/specificity were 100% concordant. Multisite sample-level genotyping accuracy was 99.2%. Across PCR and CE instruments, sample-level genotyping accuracy was 97.1%, with greater than 99% agreement for all variant-level metrics. CONCLUSIONS.­: The CFTR assay achieves 92% or higher coverage of CFTR variants in diverse populations and provides improved pan-ethnic coverage of minority subgroups of the US populace. The assay can be completed within 5 hours from DNA sample to genotype, and performance data exceed acceptance criteria for analytic metrics. This assay panel content may help address gaps in ancestry-specific CFTR genotypes while providing a streamlined procedure with rapidly generated results.

Clinically significant findings in a decade-long retrospective study of prenatal chromosomal microarray testing.

BACKGROUND: Chromosomal microarray (CMA) is commonly utilized in the obstetrics setting. CMA is recommended when one or more fetal structural abnormalities is identified. CMA is also commonly used to determine genetic etiologies for miscarriages, fetal demise, and confirming positive prenatal cell-free DNA screening results. METHODS: In this study, we retrospectively examined 523 prenatal and 319 products-of-conception (POC) CMA cases tested at Nationwide Children's Hospital from 2011 to 2020. We reviewed the referral indications, the diagnostic yield, and the reported copy number variants (CNV) findings. RESULTS: In our cohort, the diagnostic yield of clinically significant CNV findings for prenatal testing was 7.8% (n = 41/523) compared to POC testing (16.3%, n = 52/319). Abnormal ultrasound findings were the most common indication present in 81% of prenatal samples. Intrauterine fetal demise was the common indication identified in POC samples. The most common pathogenic finding observed in all samples was isolated trisomy 21, detected in seven samples. CONCLUSION: Our CMA study supports the clinical utility of prenatal CMA for clinical management and identifying genetic etiology in POC arrays. In addition, it provides insight to the spectrum of prenatal and POC CMA results as detected in an academic hospital clinical laboratory setting that serves as a reference laboratory.

Deep Brain Stimulation for the Management of AIFM1-Related Disabling Tremor: A Case Series.

The AIFM1 gene encodes a mitochondrial protein that acts as a flavin adenine dinucleotide-dependent nicotinamide adenine dinucleotide oxidase and apoptosis regulator. Monoallelic pathogenic AIFM1 variants result in a spectrum of X-linked neurological disorders, including Cowchock syndrome. Common features in Cowchock syndrome include a slowly progressive movement disorder, cerebellar ataxia, progressive sensorineural hearing loss, and sensory neuropathy. We identified a novel maternally inherited hemizygous missense AIFM1 variant, c.1369C>T p.(His457Tyr), in two brothers with clinical features consistent with Cowchock syndrome using next-generation sequencing. Both individuals had a progressive complex movement disorder phenotype, including disabling tremor poorly responsive to medications. Deep brain stimulation (DBS) of the ventral intermediate thalamic nucleus ameliorated contralateral tremor and improved their quality of life; this suggests the beneficial role for DBS in treatment-resistant tremor within AIFM1-related disorders.

Expanding the Clinical Utility of Targeted RNA Sequencing Panels beyond Gene Fusions to Complex, Intragenic Structural Rearrangements.

Gene fusions are a form of structural rearrangement well established as driver events in pediatric and adult cancers. The identification of such events holds clinical significance in the refinement, prognostication, and provision of treatment in cancer. Structural rearrangements also extend beyond fusions to include intragenic rearrangements, such as internal tandem duplications (ITDs) or exon-level deletions. These intragenic events have been increasingly implicated as cancer-promoting events. However, the detection of intragenic rearrangements may be challenging to resolve bioinformatically with short-read sequencing technologies and therefore may not be routinely assessed in panel-based testing. Within an academic clinical laboratory, over three years, a total of 608 disease-involved samples (522 hematologic malignancy, 86 solid tumors) underwent clinical testing using Anchored Multiplex PCR (AMP)-based RNA sequencing. Hematologic malignancies were evaluated using a custom Pan-Heme 154 gene panel, while solid tumors were assessed using a custom Pan-Solid 115 gene panel. Gene fusions, ITDs, and intragenic deletions were assessed for diagnostic, prognostic, or therapeutic significance. When considering gene fusions alone, we report an overall diagnostic yield of 36% (37% hematologic malignancy, 41% solid tumors). When including intragenic structural rearrangements, the overall diagnostic yield increased to 48% (48% hematologic malignancy, 45% solid tumor). We demonstrate the clinical utility of reporting structural rearrangements, including gene fusions and intragenic structural rearrangements, using an AMP-based RNA sequencing panel.

Phenotypic Spectrum in a Family Sharing a Heterozygous KCNQ3 Variant.

BACKGROUND AND PURPOSE: Mutations in KCNQ3 have classically been associated with benign familial neonatal and infantile seizures and more recently identified in patients with neurodevelopmental disorders and abnormal electroencephalogram (EEG) findings. We present 4 affected patients from a family with a pathogenic mutation in KCNQ3 with a unique constellation of clinical findings. METHODS: A family of 3 affected siblings and mother sharing a KCNQ3 pathogenic variant are described, including clinical history, genetic results, and EEG and magnetic resonance imaging (MRI) findings. RESULTS: This family shows a variety of clinical manifestations, including neonatal seizures, developmental delays, autism spectrum disorder, and anxiety. One child developed absence epilepsy, 2 children have infrequent convulsive seizures that have persisted into childhood, and their parent developed adult-onset epilepsy. An underlying c.1091G>A (R364H) variant in KCNQ3 was found in all affected individuals. CONCLUSIONS: The phenotypic variability of KCNQ3 channelopathies continues to expand as more individuals and families are described, and the variant identified in this family adds to the understanding of the manifestations of KCNQ3-related disorders.

A Decade's Experience in Pediatric Chromosomal Microarray Reveals Distinct Characteristics Across Ordering Specialties.

Chromosomal microarray (CMA) is a testing modality frequently used in pediatric patients; however, published data on its utilization are limited to the genetic setting. We performed a database search for all CMA testing performed from 2010 to 2020, and delineated the diagnostic yield based on patient characteristics, including sex, age, clinical specialty of providers, indication of testing, and pathogenic finding. The indications for testing were further categorized into Human Phenotype Ontology categories for analysis. This study included a cohort of 14,541 patients from 29 different medical specialties, of whom 30% were from the genetics clinic. The clinical indications for testing suggested that neonatology patients demonstrated the greatest involvement of multiorgan systems, involving the most Human Phenotype Ontology categories, compared with developmental behavioral pediatrics and neurology patients being the least. The top pathogenic findings for each specialty differed, likely due to the varying clinical features and indications for testing. Deletions involving the 22q11.21 locus were the top pathogenic findings for patients presenting to genetics, neonatology, cardiology, and surgery. Our data represent the largest pediatric cohort published to date. This study is the first to demonstrate the diagnostic utility of this assay for patients seen in the setting of different specialties, and it provides normative data of CMA results among a general pediatric population referred for testing because of variable clinical presentations.

Pacific Biosciences Fusion and Long Isoform Pipeline for Cancer Transcriptome-Based Resolution of Isoform Complexity.

Genomic profiling using short-read sequencing has utility in detecting disease-associated variation in both DNA and RNA. However, given the frequent occurrence of structural variation in cancer, molecular profiling using long-read sequencing improves the resolution of such events. For example, the Pacific Biosciences long-read RNA-sequencing (Iso-Seq) transcriptome protocol provides full-length isoform characterization, discernment of allelic phasing, and isoform discovery, and identifies expressed fusion partners. The Pacific Biosciences Fusion and Long Isoform Pipeline (PB_FLIP) incorporates a suite of RNA-sequencing software analysis tools and scripts to identify expressed fusion partners and isoforms. In addition, sequencing of a commercial reference (Spike-In RNA Variants) with known isoform complexity was performed and demonstrated high recall of the Iso-Seq and PB_FLIP workflow to benchmark our protocol and analysis performance. This study describes the utility of Iso-Seq and PB_FLIP analysis in improving deconvolution of complex structural variants and isoform detection within an institutional pediatric and adolescent/young adult translational cancer research cohort. The exemplar case studies demonstrate that Iso-Seq and PB_FLIP discover novel expressed fusion partners, resolve complex intragenic alterations, and discriminate between allele-specific expression profiles.

Protein Z levels in pregnant Omani women: correlation with pregnancy outcome.

Placental insufficiency resulting in fetal loss has been recognized in women with thrombophilic predisposition. Recent studies indicate that there is a high prevalence of protein Z (PZ) deficiency in patients with unexplained fetal loss. The objective of this study was to measure the PZ levels in pregnant Omani women in the first, second and third trimesters and correlate with the pregnancy outcome. The study enrolled 126 consecutive pregnant women after an informed consent prospectively. PZ was estimated in the first, second and third trimester in 15, 97 and 66 pregnant women respectively and they were followed for pregnancy outcomes including live birth, still birth, spontaneous abortion/induced abortion, maternal complications, fetal complications and health risks/complications in the newborn. The median PZ level (Mean ± SD) in the first, second and third trimester were 0.98 (1.07 ± 0.46), 1.3 (1.36 ± 0.61) and 1.44 (1.43 ± 0.69) (P < 0.05, Student's t-test, between first vs. second and first vs. third trimester). PZ deficiency defined as PZ level below 0.54 µg/ml (below 10th centile in the Omani population) was observed in 4 (4.7%) women, but interestingly all had a normal pregnancy outcome. Amongst the 43 subjects in whom paired PZ estimations were available, reducing PZ levels were observed from baseline values in 8 (33%) with normal pregnancy outcome; 5 (55%), with diabetes; 3 (50%) with hypertension and 2 (50%) with low birth weight respectively (P < 0.05, chi square test). PZ values increased progressively during the three trimesters of pregnancy. However, this increase is blunted in patients with abnormal pregnancy outcome like low birth weight babies or pregnancies associated hypertension or diabetes. Isolated PZ deficiency alone did not result in an abnormal outcome in this cohort of subjects.

Somatic variation as an incidental finding in the pediatric next-generation sequencing era.

The methodologic approach used in next-generation sequencing (NGS) affords a high depth of coverage in genomic analysis. Inherent in the nature of genomic testing, there exists potential for identifying genomic findings that are incidental or secondary to the indication for clinical testing, with the frequency dependent on the breadth of analysis and the tissue sample under study. The interpretation and management of clinically meaningful incidental genomic findings is a pressing issue particularly in the pediatric population. Our study describes a 16-mo-old male who presented with profound global delays, brain abnormality, progressive microcephaly, and growth deficiency, as well as metopic craniosynostosis. Clinical exome sequencing (ES) trio analysis revealed the presence of two variants in the proband. The first was a de novo variant in the PPP2R1A gene (c.773G > A, p.Arg258His), which is associated with autosomal dominant (AD) intellectual disability, accounting for the proband's clinical phenotype. The second was a recurrent hotspot variant in the CBL gene (c.1111T > C, p.Tyr371His), which was present at a variant allele fraction of 11%, consistent with somatic variation in the peripheral blood sample. Germline pathogenic variants in CBL are associated with AD Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia. Molecular analyses using a different tissue source, buccal epithelial cells, suggest that the CBL alteration may represent a clonal population of cells restricted to leukocytes. This report highlights the laboratory methodologic and interpretative processes and clinical considerations in the setting of acquired variation detected during clinical ES in a pediatric patient.

Pathogenic variants in SMARCA5, a chromatin remodeler, cause a range of syndromic neurodevelopmental features.

Intellectual disability encompasses a wide spectrum of neurodevelopmental disorders, with many linked genetic loci. However, the underlying molecular mechanism for more than 50% of the patients remains elusive. We describe pathogenic variants in SMARCA5, encoding the ATPase motor of the ISWI chromatin remodeler, as a cause of a previously unidentified neurodevelopmental disorder, identifying 12 individuals with de novo or dominantly segregating rare heterozygous variants. Accompanying phenotypes include mild developmental delay, frequent postnatal short stature and microcephaly, and recurrent dysmorphic features. Loss of function of the SMARCA5 Drosophila ortholog Iswi led to smaller body size, reduced sensory dendrite complexity, and tiling defects in larvae. In adult flies, Iswi neural knockdown caused decreased brain size, aberrant mushroom body morphology, and abnormal locomotor function. Iswi loss of function was rescued by wild-type but not mutant SMARCA5. Our results demonstrate that SMARCA5 pathogenic variants cause a neurodevelopmental syndrome with mild facial dysmorphia.

Saline infusion sonohysterography - an effective tool for evaluation of the endometrial cavity in women with abnormal uterine bleeding.

We evaluated the diagnostic accuracy of saline infusion sonohysterography (SISH) over transvaginal sonography (TVS) for the detection of intrauterine abnormalities using hysteroscopy as the gold standard in a retrospective study of 70 women mostly presenting with abnormal uterine bleeding. TVS was normal in 32 women, while in the others polyps or fibroids projecting into the cavity (n = 19) or a thick endometrium (n = 19) were suspected. On performing SISH the uterine cavity was found to be normal in 29 women, while 35 had suspected polyps/fibroids and 6 had other abnormalities. Hysteroscopy proved to be normal in 28 women, 35 had polyps/fibroids and 7 had other abnormalities. The sensitivity, specificity, positive and negative predictive values for TVS were 72.4%, 100%, 100% and 74%, respectively, while for SISH the corresponding figures were 91.4%, 92.6%, 89.3% and 94.1%. SISH is a simple, minimally invasive and cost-effective investigative tool enhancing the diagnostic accuracy of TVS and can be an effective screening test prior to hysteroscopy.

Erythrocyte indices, microminerals and ratios, antioxidants and lipids in Centrum Materna diet-supplemented Omani mothers.

Venous (maternal) and cord blood (neonatal) samples of Omani women who had a daily supplement of Centrum Materna multivitamin and multimineral tablet throughout pregnancy were investigated at late preterm (n=37) and at term (n=37) delivery for erythrocyte indices, micromineral, antioxidant, and lipid values. Hemoglobin (Hb), hematocrit (HCT), mean cell volume (MCV), red cell distribution width (RDW), copper (Cu), zinc (Zn), ceruloplasmin, erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD), cholesterol, apolipoprotein (apo) A-I and apo B were measured by appropriate analytical systems. Cu/zinc and Cu/ceruloplasmin ratios were calculated. The erythrocyte indices were normal in neonatal blood but showed borderline anemia in maternal blood of both groups. There were significantly decreased values of Cu (P=0.012), Zn (P=0.001), apo A-I (P=0.029), and Cu/ceruloplasmin ratio (P=0.032) in late preterm compared to term mothers. Significantly decreased values of Cu (P=0.003), ceruloplasmin (P<0.0001), apo A-I (P=0.024), and Cu/Zn ratio (P=007) were observed in late preterm relative to term neonates. Late preterm mothers were significantly younger (P=0.027) than term mothers. Maternal age correlated positively with apo A-I (r=0.424, P=0.012) and negatively with Cu/Zn ratio (r=-0.353, P=0.040). The findings suggest that with daily dietary Centrum Materna supplementation throughout pregnancy, hematological indices were maintained within normal in mothers and neonates, but the levels of microminerals and micromineral ratios were subnormal in late preterm mothers and their neonates.

Polyhydramnios. Risk factors and outcome.

OBJECTIVE: To determine the risk factors associated with polyhydramnios, and assess the maternal and perinatal outcome in these patients. METHODS: A prospective study of all deliveries complicated with polyhydramnios in Sultan Qaboos University Hospital, Sultanate of Oman between January 2005 and April 2006. Polyhydramnios was divided into mild and moderate to severe based on the amniotic fluid index values. The demographic data, antenatal complications, gestational age at delivery, mode of delivery, postpartum complications, and perinatal outcome were studied. RESULTS: A total of 2648 singleton deliveries occurred during the study period. Two hundred and eight (7.8%) women with polyhydramnios formed the study group, and 2440 women with normal amniotic fluid formed the control. Polyhydramnios was mild in 179 (86.1%) and moderate to severe in 29 (13.9%) cases. Sixty-eight (32.7%) of these pregnancies were complicated with diabetes as compared with 12.4% of the controls. Preterm delivery occurred in 16 (7.7%) cases. Cesarean delivery rate was 27.9% in the study group compared with 17.3% in the control. Major congenital anomalies were found in 2.8% of newborns compared with 1% among the controls. Eighteen babies were admitted to the special care baby unit. CONCLUSION: These data demonstrates a significant positive relation with maternal age, diabetes in pregnancy, and fetal macrosomia with polyhydramnios. Anemia during pregnancy, cesarean delivery rate, and congenital anomalies were significantly higher in the study group.

Ocular manifestations in a case of Wernicke's encephalopathy due to hyperemesis gravidarum.

Wernicke's Encephalopathy (WE) is a potentially devastating disorder, which if not diagnosed and treated at the earliest, could lead to dangerous neurological and ophthalmological complications. We report the ocular manifestations of WE in a pregnant woman with hyperemesis gravidarum.