Potassium channel-related epilepsy: Pathogenesis and clinical features.

Variants in potassium channel-related genes are one of the most important mechanisms underlying abnormal neuronal excitation and disturbances in the cellular resting membrane potential. These variants can cause different forms of epilepsy, which can seriously affect the physical and mental health of patients, especially those with refractory epilepsy or status epilepticus, which are common among pediatric patients and are potentially life-threatening. Variants in potassium ion channel-related genes have been reported in few studies; however, to our knowledge, no systematic review has been published. This study aimed to summarize the epilepsy phenotypes, functional studies, and pharmacological advances associated with different potassium channel gene variants to assist clinical practitioners and drug development teams to develop evidence-based medicine and guide research strategies. PubMed and Google Scholar were searched for relevant literature on potassium channel-related epilepsy reported in the past 5-10 years. Various common potassium ion channel gene variants can lead to heterogeneous epilepsy phenotypes, and functional effects can result from gene deletions and compound effects. Administration of select anti-seizure medications is the primary treatment for this type of epilepsy. Most patients are refractory to anti-seizure medications, and some novel anti-seizure medications have been found to improve seizures. Use of targeted drugs to correct aberrant channel function based on the type of potassium channel gene variant can be used as an evidence-based pathway to achieve precise and individualized treatment for children with epilepsy. PLAIN LANGUAGE SUMMARY: In this article, the pathogenesis and clinical characteristics of epilepsy caused by different types of potassium channel gene variants are reviewed in the light of the latest research literature at home and abroad, with the expectation of providing a certain theoretical basis for the diagnosis and treatment of children with this type of disease.

Functional characterization of HNF4A gene variants identify promoter and cell line specific transactivation effects.

Hepatocyte nuclear factor-4 alpha (HNF-4A) regulates genes with roles in glucose metabolism and ß-cell development. Although pathogenic HNF4A variants are commonly associated with maturity-onset diabetes of the young (MODY1; HNF4A-MODY), rare phenotypes also include hyperinsulinemic hypoglycemia, renal Fanconi syndrome and liver disease. While the association of rare functionally damaging HNF1A variants with HNF1A-MODY and type 2 diabetes is well established owing to robust functional assays, the impact of HNF4A variants on HNF-4A transactivation in tissues including the liver and kidney is less known, due to lack of similar assays. Our aim was to investigate the functional effects of seven HNF4A variants, located in the HNF-4A DNA binding domain and associated with different clinical phenotypes, by various functional assays and cell lines (transactivation, DNA binding, protein expression, nuclear localization) and in silico protein structure analyses. Variants R85W, S87N and R89W demonstrated reduced DNA binding to the consensus HNF-4A binding elements in the HNF1A promoter (35, 13 and 9%, respectively) and the G6PC promoter (R85W ~10%). While reduced transactivation on the G6PC promoter in HepG2 cells was shown for S87N (33%), R89W (65%) and R136W (35%), increased transactivation by R85W and R85Q was confirmed using several combinations of target promoters and cell lines. R89W showed reduced nuclear levels. In silico analyses supported variant induced structural impact. Our study indicates that cell line specific functional investigations are important to better understand HNF4A-MODY genotype-phenotype correlations, as our data supports ACMG/AMP interpretations of loss-of-function variants and propose assay-specific HNF4A control variants for future functional investigations.

Variant functional assessment in Drosophila by overexpression: what can we learn?

The last decade has been highlighted by the increased use of next-generation DNA sequencing technology to identify novel human disease genes. A critical downstream part of this process is assigning function to a candidate gene variant. Functional studies in Drosophila melanogaster, the common fruit fly, have made a prominent contribution in annotating variant impact in an in vivo system. The use of patient-derived knock-in flies or rescue-based, "humanization", approaches are novel and valuable strategies in variant testing but have been recently widely reviewed. An often-overlooked strategy for determining variant impact has been GAL4/upstream activation sequence-mediated tissue-defined overexpression in Drosophila. This mini-review will summarize the recent contribution of ectopic overexpression of human reference and variant cDNA in Drosophila to assess variant function, interpret the consequence of the variant, and in some cases infer biological mechanisms.

Combining a prioritization strategy and functional studies nominates 5'UTR variants underlying inherited retinal disease.

BACKGROUND: 5' untranslated regions (5'UTRs) are essential modulators of protein translation. Predicting the impact of 5'UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5'UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). METHODS: We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5'UTRs by different whole exome sequencing (WES) kits. The selected 5'UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5'UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches. RESULTS: Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5'UTR compared to that of their canonical isoform. Depending on the probe design, 3-20% of IRD genes have 5'UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5'UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. CONCLUSIONS: This study demonstrates the importance of 5'UTR variants implicated in IRDs and provides a systematic approach for 5'UTR annotation and validation that is applicable to other inherited diseases.

Recessive SERPING1 Variant Leads to Kinin-Kallikrein System Control Failure in a Consanguineous Brazilian Family with Hereditary Angioedema.

Background: Hereditary angioedema (HAE) is a severe and potentially life-threatening disease. The most common forms are caused by variants in SERPING1, resulting in C1-inhibitor (C1-INH) deficiency (HAE-C1-INH). C1-INH is a serine protease inhibitor (SERPIN) that regulates multiple proteases pathways, including the kallikrein-kinin system (KKS) and its complement. In HAE-C1-INH patients, C1-INH deficiencies affect KKS control, resulting in the development of kallikrein activity in plasma and the subsequent release of bradykinin (BK). While the overwhelming majority of disease-causing SERPING1 variants are dominant, very few recessive variants have been described. We present a large Brazilian HAE-C1-INH family with a recessive form of HAE-C1-INH. Methods: Blood samples of family members were investigated for protein levels of C1-INH, C4, C1q, and C1-INH function. The SERPING1 gene was sequenced. Results: In two severely affected sisters, we identified a homozygous missense variant in SERPING1 (NM_000062.3:c.964G>A;p.Val322Met). Fourteen family members were asymptomatic heterozygous carriers of the variant. Data regarding C1-INH function in the plasma showed that homozygous p.Val322Met strongly impacts C1-INH function to inhibit C1s and kallikrein (PKa). When heterozygously expressed, it affects the C1-INH control of C1s more than that of PKa. Conclusions: These studies of the variant's effects on the structure-function relationship reinforce prior observations suggesting that C1-INH deficiency is a conformational disease.

Erratum: Functional and clinical analysis of five EDA variants associated with ectodermal dysplasia but with a hard-to-predict significance.

[This corrects the article DOI: 10.3389/fgene.2022.934395.].

Advanced Optical Microscopy: Unveiling Functional Insights Regarding a Novel PPP2R1A Variant and Its Unreported Phenotype.

The number of genes implicated in neurodevelopmental conditions is rapidly growing. Recently, variants in PPP2R1A have been associated with syndromic intellectual disability and a consistent, but still expanding, phenotype. The PPP2R1A gene encodes a protein subunit of the serine/threonine protein phosphatase 2A enzyme, which plays a critical role in cellular function. We report an individual showing pontocerebellar hypoplasia (PCH), microcephaly, optic and peripheral nerve abnormalities, and an absence of typical features like epilepsy and an abnormal corpus callosum. He bears an unreported variant in an atypical region of PPP2R1A. In silico studies, functional analysis using immunofluorescence, and super-resolution microscopy techniques were performed to investigate the pathogenicity of the variant. This analysis involved a comparative analysis of the patient's fibroblasts with both healthy control cells and cells from an individual with the previously described phenotype. The results showed reduced expression of PPP2R1A and the presence of aberrant protein aggregates in the patient's fibroblasts, supporting the pathogenicity of the variant. These findings suggest a potential association between PPP2R1A variants and PCH, expanding the clinical spectrum of PPP2R1A-related neurodevelopmental disorder. Further studies and descriptions of additional patients are needed to fully understand the genotype-phenotype correlation and the underlying mechanisms of this novel phenotype.

A functional mechanism for a non-coding variant near AGTR2 associated with risk for preterm birth.

BACKGROUND: Preterm birth (PTB), defined as delivery before 37 gestational weeks, imposes significant public health burdens. A recent maternal genome-wide association study of spontaneous PTB identified a noncoding locus near the angiotensin II receptor type 2 (AGTR2) gene. Genotype-Tissue Expression data revealed that alleles associated with decreased AGTR2 expression in the uterus were linked to an increased risk of PTB and shortened gestational duration. We hypothesized that a causative variant in this locus modifies AGTR2 expression by altering transcription factor (TF) binding. METHODS: To investigate this hypothesis, we performed bioinformatics analyses and functional characterizations at the implicated locus. Potential causal single nucleotide polymorphisms (SNPs) were prioritized, and allele-dependent binding of TFs was predicted. Reporter assays were employed to assess the enhancer activity of the top PTB-associated non-coding variant, rs7889204, and its impact on TF binding. RESULTS: Our analyses revealed that rs7889204, a top PTB-associated non-coding genetic variant is one of the strongest eQTLs for the AGTR2 gene in uterine tissue samples. We observed differential binding of CEBPB (CCAAT enhancer binding protein beta) and HOXA10 (homeobox A10) to the alleles of rs7889204. Reporter assays demonstrated decreased enhancer activity for the rs7889204 risk "C" allele. CONCLUSION: Collectively, these results demonstrate that decreased AGTR2 expression caused by reduced transcription factor binding increases the risk for PTB and suggest that enhancing AGTR2 activity may be a preventative measure in reducing PTB risk.

Structural and functional insights into a novel homozygous missense pathogenic variant in CUL7 identified in consanguineous Pakistani family.

3M syndrome is a rare genetic familial disorder characterized by short stature, growth retardation, facial dysmorphism, skeletal abnormalities, fleshy protruding heels, and normal intelligence, caused by mutations in the CUL7, OBSL1 and CCDC8 genes. In the present study, a novel homozygous missense variant of CUL7 (NP_001161842.1, c.4493T > C, p.L1498P) has been identified in a consanguineous Pakistani family by whole exome sequencing. In silico structural evaluation, molecular docking and simulation studies of mutant CUL7 provides substantial evidence about its crucial role in the progression of discussed ailment. The newly discovered variant significantly altered the protein's three dimensional structure, leading to abnormal interaction with binding proteins. This computational and experimental investigation provides useful information to drug developers for the synthesis of novel therapeutics against the discussed ailment.Communicated by Ramaswamy H. Sarma.

Functional characterization of a novel PRRT2 variant found in a Portuguese patient with hemiplegic migraine.

Familial hemiplegic migraine (FHM) is a rare autosomal-dominant form of migraine with aura. Three disease-causing genes have been identified for FHM: CACNA1A, ATP1A2 and SCN1A. However, not all families are linked to one of these three genes.PRRT2 variants were also commonly associated with HM symptoms; therefore, PRRT2 is hypothesized as the fourth gene causing FHM. PRRT2 plays an important role in neuronal migration, spinogenesis, and synapse mechanisms during development and calcium-dependent neurotransmitter release. We performed exome sequencing to unravel the genetic cause of migraine in one family, and a novel PRRT2 variant (c.938C > T;p.Ala313Val) was identified with further functional studies to confirm its pathogenicity. PRRT2-A313V reduced protein stability, led to protein premature degradation by the proteasome and altered the subcellular localization of PRRT2 from the plasma membrane (PM) to the cytoplasm. We identified and characterized for the first time in a Portuguese patient, a novel heterozygous missense variant in PRRT2 associated with HM symptoms. We suggest that PRRT2 should be included in the diagnosis of HM.

Carriers of the p.P522R variant in PLCγ2 have a slightly more responsive immune system.

BACKGROUND: The rs72824905 single-nucleotide polymorphism in the PLCG2 gene, encoding the p.P522R residue change in Phospholipase C gamma 2 (PLCγ2), associates with protection against several dementia subtypes and with increased likelihood of longevity. Cell lines and animal models indicated that p.P522R is a functional hypermorph. We aimed to confirm this in human circulating peripheral immune cells. METHODS: We compared effects of p.P522R on immune system function between carriers and non-carriers (aged 59-103y), using in-depth immunophenotyping, functional B-cell and myeloid cell assays, and in vivo SARS-CoV-2 vaccination. RESULTS: In line with expectations, p.P522R impacts immune cell function only slightly, but it does so across a wide array of immune cell types. Upon B-cell stimulation, we observed increased PLCγ2 phosphorylation and calcium release, suggesting increased B-cell sensitivity upon antigen recognition. Further, p.P522R-carriers had higher numbers of CD20++CD21-CD24+ naive B cells and IgG1+ memory B cells. In myeloid cells, normalized ROS production was higher upon PLCγ2-dependent stimulation. On classical monocytes, CD33 levels were elevated. Furthermore, carriers expressed lower levels of allergy-related FcεRI on several immune cell subsets. Nevertheless, carriers and non-carriers had similar serological responses to SARS-CoV-2 vaccination. CONCLUSION: The immune system from p.P522R-carriers is slightly more responsive to stimulation than in non-carriers.

A rabies virus-based toolkit for efficient retrograde labeling and monosynaptic tracing.

Analyzing the structure and function of the brain's neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases. Recombinant rabies viral vectors allow for the retrograde labeling of projection neurons and cell type-specific trans-monosynaptic tracing, making these vectors powerful candidates for the dissection of synaptic inputs. Although several attenuated rabies viral vectors have been developed, their application in studies of functional networks is hindered by the long preparation cycle and low yield of these vectors. To overcome these limitations, we developed an improved production system for the rapid rescue and preparation of a high-titer CVS-N2c-ΔG virus. Our results showed that the new CVS-N2c-ΔG-based toolkit performed remarkably: (1) N2cG-coated CVS-N2c-ΔG allowed for efficient retrograde access to projection neurons that were unaddressed by rAAV9-Retro, and the efficiency was six times higher than that of rAAV9-Retro; (2) the trans-monosynaptic efficiency of oG-mediated CVS-N2c-ΔG was 2-3 times higher than that of oG-mediated SAD-B19-ΔG; (3) CVS-N2c-ΔG could delivery modified genes for neural activity monitoring, and the time window during which this was maintained was 3 weeks; and (4) CVS-N2c-ΔG could express sufficient recombinases for efficient transgene recombination. These findings demonstrate that new CVS-N2c-ΔG-based toolkit may serve as a versatile tool for structural and functional studies of neural circuits.

Mild Neurological Phenotype Associated with Hypomorphic Variants in the Ataxia-Telangiectasia Mutated Gene.

Background: Ataxia-telangiectasia (A-T) is a progressive multisystemic neurodegenerative disease. The phenotypic spectrum includes conditions (variant A-T) with mild, late-onset, and atypical clinical presentations characterized by the prevalence of dyskinetic rather than ataxic features. Cases: We describe the clinical presentations of 3 siblings with early-onset truncal ataxia without obvious neurological deterioration or biological markers of classic A-T phenotype. We performed functional and genetic evaluation of 3 siblings with very mild neurological phenotype. Genetic evaluation with a next-generation sequencing panel for genes causative of cerebellar ataxia detected 2 known ATM gene variants, missense c.9023G>A p.(Arg3008His), and leaky splicing c.1066-6T>G variants. Functional studies showed mildly reduced ATM expression and residual kinase activity in the probands compared with healthy controls. Conclusions: These results suggest the importance of investigating ATM variants even in the presence of clinical and biological atypical cases to ensure specific therapeutic regimens and oncological surveillance in these patients.

Detection of novel variant and functional study in a Chinese family with nonsyndromic oligodontia.

OBJECTIVES: To investigate the pathogenic gene of a patient with nonsyndromic oligodontia, and analyze its possible pathogenic mechanism. SUBJECTS AND METHODS: The variant was detected by whole exome sequencing (WES) and Sanger sequencing in a family with oligodontia. Bioinformatic and structural analyses were used to analyze variant. Functional studies including western blotting and immunofluorescent analyses and luciferase reporter assay were conducted to explore the functional effects. RESULTS: We identified a novel frameshift variant of PAX9 (c.491-510delGCCCT-ATCACGGCGGCGGCC, p.P165Qfs*145) outside the DNA-binding domain causing an autosomal-dominant nonsyndromic oligodontia in a Chinese family. Bioinformatic and structural analyses revealed that the variant is pathogenic and conserved evolutionarily, and the changes might affect protein stability or folding. Functional studies demonstrate dramatically reduced ability in activating transcription activity of BMP4 promoter and a marked decrease in protein production, as evaluated by western blotting and immunofluorescent analyses. CONCLUSIONS: We found a novel frameshift variant of PAX9 causing nonsyndromic oligodontia in a Chinese family. Our findings indicate that frameshift variants cause loss of function of PAX9 protein during the patterning of the dentition and the subsequent tooth agenesis, providing new molecular insights into the role of frameshift variant of PAX9 and broaden the pathogenic spectrum of PAX9 variants.

Concise Review: Stem Cell Models of SCN1A-Related Encephalopathies-Current Perspective and Future Therapies.

Mutations in the SCN1A gene can cause a variety of phenotypes, ranging from mild forms, such as febrile seizures and generalized epilepsy with febrile seizures plus, to severe, such as Dravet and non-Dravet developmental epileptic encephalopathies. Until now, more than two thousand pathogenic variants of the SCN1A gene have been identified and different pathogenic mechanisms (loss vs. gain of function) described, but the precise molecular mechanisms responsible for the deficits exhibited by patients are not fully elucidated. Additionally, the phenotypic variability proves the involvement of other genetic factors in its final expression. This is the reason why animal models and cell line models used to explore the molecular pathology of SCN1A-related disorders are only of limited use. The results of studies based on such models cannot be directly translated to affected individuals because they do not address each patient's unique genetic background. The generation of functional neurons and glia for patient-derived iPSCs, together with the generation of isogenic controls using CRISPR/Cas technology, and finally, the 3D brain organoid models, seem to be a good way to solve this problem. Here, we review SCN1A-related encephalopathies, as well as the stem cell models used to explore their molecular basis.

Development of a clinically validated in vitro functional assay to assess pathogenicity of novel GAA variants in patients with Pompe disease identified via newborn screening.

Purpose: The addition of Pompe disease (Glycogen Storage Disease Type II) to the Recommended Uniform Screening Panel in the United States has led to an increase in the number of variants of uncertain significance (VUS) and novel variants identified in the GAA gene. This presents a diagnostic challenge, especially in the setting of late-onset Pompe disease when symptoms are rarely apparent at birth. There is an unmet need for validated functional studies to aid in classification of GAA variants. Methods: We developed an in vitro mammalian cell expression and functional analysis system based on guidelines established by the Clinical Genome Resource (ClinGen) Sequence Variant Interpretation Working Group for PS3/BS3. We validated the assay with 12 control variants and subsequently analyzed eight VUS or novel variants in GAA identified in patients with a positive newborn screen for Pompe disease without phenotypic evidence of infantile-onset disease. Results: The control variants were analyzed in our expression system and an activity range was established. The pathogenic controls had GAA activity between 0% and 11% of normal. The benign or likely benign controls had an activity range of 54%-100%. The pseudodeficiency variant had activity of 17%. These ranges were then applied to the variants selected for functional studies. Using the threshold of <11%, we were able to apply PS3_ supporting to classify two variants as likely pathogenic (c.316C > T and c.1103G > A) and provide further evidence to support the classification of likely pathogenic for two variants (c.1721T > C and c.1048G > A). One variant (c.1123C > T) was able to be reclassified based on other supporting evidence. We were unable to reclassify three variants (c.664G > A, c.2450A > G, and c.1378G > A) due to insufficient or conflicting evidence. Conclusion: We investigated eight GAA variants as proof of concept using our validated and reproducible in vitro expression and functional analysis system. While additional work is needed to further refine our system with additional controls and different variant types in order to apply the PS3/BS3 criteria at a higher level, this tool can be utilized for variant classification to meet the growing need for novel GAA variant classification in the era of newborn screening for Pompe disease.

MKRN3 role in regulating pubertal onset: the state of art of functional studies.

Puberty is a critical process characterized by several physical and psychological changes that culminate in the achievement of sexual maturation and fertility. The onset of puberty depends on several incompletely understood mechanisms that certainly involve gonadotropin-releasing hormone (GnRH) and its effects on the pituitary gland. The role of makorin ring finger protein 3 (MKRN3) in the regulation of pubertal timing was revealed when loss-of-function mutations were identified in patients with central precocious puberty (CPP), which to date, represent the most commonly known genetic cause of this condition. The MKRN3 gene showed ubiquitous expression in tissues from a broad spectrum of species, suggesting an important cellular role. Its involvement in the initiation of puberty and endocrine functions has just begun to be studied. This review discusses some of the recent approaches developed to predict MKRN3 functions and its involvement in pubertal development.

Imaging Techniques: Essential Tools for the Study of SARS-CoV-2 Infection.

The world has seen the emergence of a new virus in 2019, SARS-CoV-2, causing the COVID-19 pandemic and millions of deaths worldwide. Microscopy can be much more informative than conventional detection methods such as RT-PCR. This review aims to present the up-to-date microscopy observations in patients, the in vitro studies of the virus and viral proteins and their interaction with their host, discuss the microscopy techniques for detection and study of SARS-CoV-2, and summarize the reagents used for SARS-CoV-2 detection. From basic fluorescence microscopy to high resolution techniques and combined technologies, this article shows the power and the potential of microscopy techniques, especially in the field of virology.

Functional and clinical analysis of five EDA variants associated with ectodermal dysplasia but with a hard-to-predict significance.

Deficiency of ectodysplasin A1 (EDA1) due to variants of the gene EDA causes X-linked hypohidrotic ectodermal dysplasia (XLHED), a rare genetic condition characterized by abnormal development of ectodermal structures. XLHED is defined by the triad of hypotrichosis, hypo- or anhidrosis, and hypo- or anodontia. Anhidrosis may lead to life-threatening hyperthermia. A definite genetic diagnosis is, thus, important for the patients' management and amenability to a novel prenatal treatment option. Here, we describe five familial EDA variants segregating with the disease in three families, for which different prediction tools yielded discordant results with respect to their significance. Functional properties in vitro and levels of circulating serum EDA were compared with phenotypic data on skin, hair, eyes, teeth, and sweat glands. EDA1-Gly176Val, although associated with relevant hypohidrosis, still bound to the EDA receptor (EDAR). Subjects with EDA1-Pro389LeufsX27, -Ter392GlnfsX30, -Ser125Cys, and an EDA1 splice variant (c.924+7A > G) showed complete absence of pilocarpine-induced sweating. EDA1-Pro389LeufsX27 was incapable of binding to EDAR and undetectable in serum. EDA1-Ter392GlnfsX30, produced in much lower amounts than wild-type EDA1, could still bind to EDAR, and so did EDA1-Ser125Cys that was, however, undetectable in serum. The EDA splice variant c.924+7A > G resulted experimentally in a mix of wild-type EDA1 and EDA molecules truncated in the middle of the receptor-binding domain, with reduced EDA serum concentration. Thus, in vitro assays reflected the clinical phenotype in two of these difficult cases, but underestimated it in three others. Absence of circulating EDA seems to predict the full-blown phenotype of XLHED, while residual EDA levels may also be found in anhidrotic patients. This indicates that unborn subjects carrying variants of uncertain significance could benefit from an upcoming prenatal medical treatment even if circulating EDA levels or tests in vitro suggest residual EDA1 activity.