Pathogenic variants in HGF give rise to childhood-to-late onset primary lymphoedema by loss of function.

Developmental and functional defects in the lymphatic system are responsible for primary lymphoedema (PL). PL is a chronic debilitating disease caused by increased accumulation of interstitial fluid, predisposing to inflammation, infections and fibrosis. There is no cure, only symptomatic treatment is available. Thirty-two genes or loci have been linked to PL, and another 22 are suggested, including Hepatocyte Growth Factor (HGF). We searched for HGF variants in 770 index patients from the Brussels PL cohort. We identified ten variants predicted to cause HGF loss-of-function (six nonsense, two frameshifts, and two splice-site changes; 1.3% of our cohort), and 14 missense variants predicted to be pathogenic in 17 families (2.21%). We studied co-segregation within families, mRNA stability for non-sense variants, and in vitro functional effects of the missense variants. Analyses of the mRNA of patient cells revealed degradation of the nonsense mutant allele. Reduced protein secretion was detected for nine of the 14 missense variants expressed in COS-7 cells. Stimulation of lymphatic endothelial cells with these 14 HGF variant proteins resulted in decreased activation of the downstream targets AKT and ERK1/2 for three of them. Clinically, HGF-associated PL was diverse, but predominantly bilateral in the lower limbs with onset varying from early childhood to adulthood. Finally, aggregation study in a second independent cohort underscored that rare likely pathogenic variants in HGF explain about 2% of PL. Therefore, HGF signalling seems crucial for lymphatic development and/or maintenance in human beings and HGF should be included in diagnostic genetic screens for PL.

Phenotypic spectrum of patients with Poretti-Boltshauser syndrome: Patient report of antenatal ventriculomegaly and esophageal atresia.

Poretti-Boltshauser syndrome (PTBHS) is an autosomal recessive disorder characterized by cerebellar dysplasia with cysts and an abnormal shape of the fourth ventricle on neuroimaging, due to pathogenic variants in the LAMA1 gene. The clinical spectrum mainly consists of neurological and ophthalmological manifestations, including non-progressive cerebellar ataxia, oculomotor apraxia, language impairment, intellectual disability, high myopia, abnormal eye movements and retinal dystrophy. We report a patient presenting with ventriculomegaly on antenatal neuroimaging and a neonatal diagnosis of Type III esophageal atresia. She subsequently developed severe myopia and strabismus with retinal dystrophy, mild developmental delay, and cerebellar dysplasia. Genetic investigations confirmed PTBHS. This report confirms previous reports of antenatal ventriculomegaly in PTBHS patients and documents a so far unreported occurrence of esophageal atresia in PTBHS. We additionally gathered phenotype and genotype descriptions of published cases in an effort to better define the spectrum of PTBHS.

Wieacker-Wolff syndrome, a distinctive phenotype of arthrogryposis multiplex congenita caused by a "de novo" ZC4H2 gene partial deletion.

Unusual fetal arthrogryposis on ultrasound should draw attention to look for additional lower limb anomalies. Precise genetic counseling may be obtained from deletion on Xq11.2 as for ZC4H2 gene sequencing diagnostic for Wieacker-Wolff syndrome.

Performance and Diagnostic Value of Genome-Wide Noninvasive Prenatal Testing in Multiple Gestations.

OBJECTIVE: To evaluate the accuracy and diagnostic value of genome-wide noninvasive prenatal testing (NIPT) for the detection of fetal aneuploidies in multiple gestations, with a focus on dichorionic-diamniotic twin pregnancies. METHODS: We performed a retrospective cohort study including data from pregnant women with a twin or higher-order gestation who underwent genome-wide NIPT at one of the eight Belgian genetic centers between November 1, 2013, and March 1, 2020. Chorionicity and amnionicity were determined by ultrasonography. Follow-up invasive testing was carried out in the event of positive NIPT results. Sensitivity and specificity were calculated for the detection of trisomy 21, 18, and 13 in the dichorionic-diamniotic twin cohort. RESULTS: Unique NIPT analyses were performed for 4,150 pregnant women with a multiple gestation and an additional 767 with vanishing gestations. The failure rate in multiple gestations excluding vanishing gestations ranged from 0% to 11.7% among the different genetic centers. Overall, the failure rate was 4.8%, which could be reduced to 1.2% after single resampling. There were no common fetal trisomies detected among the 86 monochorionic-monoamniotic and 25 triplet cases. Two monochorionic-diamniotic twins had an NIPT result indicative of a trisomy 21, which was confirmed in both fetuses. Among 2,716 dichorionic-diamniotic twin gestations, a sensitivity of 100% (95% CI 74.12-100%) and a specificity of 100% (95% CI 99.86-100%) was reached for trisomy 21 (n=12). For trisomy 18 (n=3), the respective values were 75% (95% CI 30.06-95.44%) sensitivity and 100% (95% CI 99.86-100%) specificity, and for trisomy 13 (n=2), 100% (95% CI 20.65-100%) sensitivity and 99.96% (95% CI 99.79-99.99%) specificity. In the vanishing gestation group, 28 NIPT results were positive for trisomy 21, 18, or 13, with only five confirmed trisomies. CONCLUSION: Genome-wide NIPT performed accurately for detection of aneuploidy in dichorionic-diamniotic twin gestations.

Antenatal diagnosis of CHARGE syndrome: Prenatal ultrasound findings and crucial role of fetal dysmorphic signs. About a series of 10 cases and review of literature.

Although the prognosis of CHARGE syndrome can be highly variable from mild until severe, final diagnosis is difficult to establish in utero. The aim of our study is to compare antenatal and postnatal findings in a retrospective cohort of 10 successive patients with a positive CHD7 gene variant in order to identify the specific prenatal features for CHARGE syndrome diagnosis. Fetal ultrasound, follow-up and supplementary investigations are collected and compared to postnatal findings. Congenital heart defect (7/10), choanal atresia (7/10) and tracheoesophageal atresia (4/10) are the most frequent fetal anomalies found. Inner and external ear anomalies appear as the keystone (constant features) for prenatal diagnosis of CHARGE syndrome in fetuses with multiple anomalies and normal microarray karyotype. External ear malformations are identified in all cases by 3D ultrasound when carefully evaluated. MRI and temporal bone CT-Scan are second line useful tools to assess the diagnosis when looking for semicircular canal agenesis, arhinencephaly and/or choanal atresia. Before availability of prenatal exome sequencing in clinical routine, present findings lead to the recommendation that fetuses, with congenital heart defect (mainly septal and conotruncal), cleft lip/palate or unexplained polyhydramnios should carefully be screened for clues suggesting CHARGE syndrome using 2D and 3D ultrasound, MRI and temporal bone CT-Scan. When CHARGE syndrome is suspected with normal molecular karyotype, CHD7 gene sequencing must be offered.

Outcome of publicly funded nationwide first-tier noninvasive prenatal screening.

PURPOSE: Noninvasive prenatal screening (NIPS) using cell-free DNA has transformed prenatal care. Belgium was the first country to implement and fully reimburse NIPS as a first-tier screening test offered to all pregnant women. A consortium consisting of all Belgian genetic centers report the outcome of two years genome-wide NIPS implementation. METHODS: The performance for the common trisomies and for secondary findings was evaluated based on 153,575 genome-wide NIP tests. Furthermore, the evolution of the number of invasive tests and the incidence of Down syndrome live births was registered. RESULTS: Trisomies 21, 18, and 13 were detected in respectively 0.32%, 0.07%, and 0.06% of cases, with overall positive predictive values (PPVs) of 92.4%, 84.6%, and 43.9%. Rare autosomal trisomies and fetal segmental imbalances were detected in respectively 0.23% and 0.07% of cases with PPVs of 4.1% and 47%. The number of invasive obstetric procedures decreased by 52%. The number of trisomy 21 live births dropped to 0.04%. CONCLUSION: Expanding the scope of NIPS beyond trisomy 21 fetal screening allows the implementation of personalized genomic medicine for the obstetric population. This genome-wide NIPS approach has been embedded successfully in prenatal genetic care in Belgium and might serve as a framework for other countries offering NIPS.

Defining a Centiloid scale threshold predicting long-term progression to dementia in patients attending the memory clinic: an [18F] flutemetamol amyloid PET study.

PURPOSE: To evaluate cerebral amyloid-ß(Aß) pathology in older adults with cognitive complaints, visual assessment of PET images is approved as the routine method for image interpretation. In research studies however, Aß-PET semi-quantitative measures are associated with greater risk of progression to dementia; but until recently, these measures lacked standardization. Therefore, the Centiloid scale, providing standardized Aß-PET semi-quantitation, was recently validated. We aimed to determine the predictive values of visual assessments and Centiloids in non-demented patients, using long-term progression to dementia as our standard of truth. METHODS: One hundred sixty non-demented participants (age, 54-86) were enrolled in a monocentric [18F] flutemetamol Aß-PET study. Flutemetamol images were interpreted visually following the manufacturers recommendations. SUVr values were converted to the Centiloid scale using the GAAIN guidelines. Ninety-eight persons were followed until dementia diagnosis or were clinically stable for a median of 6 years (min = 4.0; max = 8.0). Twenty-five patients with short follow-up (median = 2.0 years; min = 0.8; max = 3.9) and 37 patients with no follow-up were excluded. We computed ROC curves predicting subsequent dementia using baseline PET data and calculated negative (NPV) and positive (PPV) predictive values. RESULTS: In the 98 participants with long follow-up, Centiloid = 26 provided the highest overall predictive value = 87% (NPV = 85%, PPV = 88%). Visual assessment corresponded to Centiloid = 40, which predicted dementia with an overall predictive value = 86% (NPV = 81%, PPV = 92%). Inclusion of the 25 patients who only had a 2-year follow-up decreased the PPV = 67% (NPV = 88%), reflecting the many positive cases that did not progress to dementia after short follow-ups. CONCLUSION: A Centiloid threshold = 26 optimally predicts progression to dementia 6 years after PET. Visual assessment provides similar predictive value, with higher specificity and lower sensitivity. TRIAL REGISTRATION: Eudra-CT number: 2011-001756-12.

Novel RPL13 Variants and Variable Clinical Expressivity in a Human Ribosomopathy With Spondyloepimetaphyseal Dysplasia.

Spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of disorders with variable growth failure and skeletal impairments affecting the spine and long bone epiphyses and metaphyses. Here we report on four unrelated families with SEMD in which we identified two monoallelic missense variants and one monoallelic splice site variant in RPL13, encoding the ribosomal protein eL13. In two out of four families, we observed autosomal dominant inheritance with incomplete penetrance and variable clinical expressivity; the phenotypes of the mutation-positive subjects ranged from normal height with or without hip dysplasia to severe SEMD with severe short stature and marked skeletal dysplasia. In vitro studies on patient-derived dermal fibroblasts harboring RPL13 missense mutations demonstrated normal eL13 expression, with proper subcellular localization but reduced colocalization with eL28 (p < 0.001). Cellular functional defects in fibroblasts from mutation-positive subjects indicated a significant increase in the ratio of 60S subunits to 80S ribosomes (p = 0.007) and attenuated global translation (p = 0.017). In line with the human phenotype, our rpl13 mutant zebrafish model, generated by CRISPR-Cas9 editing, showed cartilage deformities at embryonic and juvenile stages. These findings extend the genetic spectrum of RPL13 mutations causing this novel human ribosomopathy with variable skeletal features. Our study underscores for the first time incomplete penetrance and broad phenotypic variability in SEMD-RPL13 type and confirms impaired ribosomal function. Furthermore, the newly generated rpl13 mutant zebrafish model corroborates the role of eL13 in skeletogenesis. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Expanding the genotypic and phenotypic spectrum of severe serine biosynthesis disorders.

Serine biosynthesis disorders comprise a spectrum of very rare autosomal recessive inborn errors of metabolism with wide phenotypic variability. Neu-Laxova syndrome represents the most severe expression and is characterized by multiple congenital anomalies and pre- or perinatal lethality. Here, we present the mutation spectrum and a detailed phenotypic analysis in 15 unrelated families with severe types of serine biosynthesis disorders. We identified likely disease-causing variants in the PHGDH and PSAT1 genes, several of which have not been reported previously. Phenotype analysis and a comprehensive review of the literature corroborates the evidence that serine biosynthesis disorders represent a continuum with varying degrees of phenotypic expression and suggest that even gradual differences at the severe end of the spectrum may be correlated with particular genotypes. We postulate that the individual residual enzyme activity of mutant proteins is the major determinant of the phenotypic variability, but further functional studies are needed to explore effects at the enzyme protein level.

Phenotype description in KIF5C gene hot-spot mutations responsible for malformations of cortical development (MCD).

Malformations of cortical development (MCD) represent a large group of brain cortical anomalies characterized by distinctive MRI findings. This 'radiologically-based' classification required re evaluation over time on identified underlying mechanisms (cytogenetic and/or molecular). The understanding of genotype findings (nature of cytogenetic/molecular mutation, cellular pathways consequences, timing, …) draw line of evidence on these distinctive group of conditions whereas sometimes precise and constant recurrent genotype/phenotype correlation may not be present. The clinical diagnosis of MCD is often difficult due to variability and rarity of individual types of malformations. Recent studies have established a relationship between lissencephaly and pathogenic variants in genes involved in the kinesin/tubulin pathways, as the KIF5C gene. Pathogenic variants in the KIF5C gene are a more recently discovered cause of severe developmental delay with epilepsy, characterized by specific malformation of cortical development such as pachygyria. Only seven children have been described to date. We report the natural history of a sixteen years old patient identified carrier of a KIF5C gene mutation who developed infantile epilepsy. We then gather phenotype description and molecular results of all reported patients so far in order to better define this entity.

Prenatally detected copy number variants in a national cohort: A postnatal follow-up study.

OBJECTIVE: Belgian genetic centers established a database containing data on all chromosomal microarrays performed in a prenatal context. A study was initiated to evaluate postnatal development in children diagnosed prenatally with a non-benign copy number variant (CNV). METHODS: All children diagnosed with a prenatally detected non-benign CNV in a Belgian genetic center between May 2013 and February 2015 were included in the patient population. The control population consisted of children who had undergone an invasive procedure during pregnancy, with no or only benign CNVs. Child development was evaluated at 36 months using three (3) questionnaires: Ages and Stages Questionnaire Third edition, Ages and Stages Questionnaire Social-Emotional Second Edition and a general questionnaire. RESULTS: A significant difference in communication and personal-social development was detected between children with a reported susceptibility CNV and both children with an unreported susceptibility CNV and the control population. The outcome of children with a particular CNV is discussed in a case-by-case manner. CONCLUSION: Our postnatal follow-up project of children with a prenatally detected non-benign CNV is the first nationwide project of its kind. A higher number of cases for each CNV category is however needed to confirm our findings.

Null variants and deletions in BRWD3 cause an X-linked syndrome of mild-moderate intellectual disability, macrocephaly, and obesity: A series of 17 patients.

BRWD3 has been described as a cause of X-linked intellectual disability, but relatively little is known about the specific phenotype. We report the largest BRWD3 patient series to date, comprising 17 males with 12 distinct null variants and 2 partial gene deletions. All patients presented with intellectual disability, which was classified as moderate (65%) or mild (35%). Behavioral issues were present in 75% of patients, including aggressive behavior, attention deficit/hyperactivity and/or autistic spectrum disorders. Mean head circumference was +2.8 SD (2.8 standard deviations above the mean), and mean BMI was +2.0 SD (in the context of a mean height of +1.3 SD), indicating a predominant macrocephaly/obesity phenotype. Shared facial features included a tall chin, prognathism, broad forehead, and prominent supraorbital ridge. Additional features, reported in a minority (<30%) of patients included cryptorchidism, neonatal hypotonia, and small joint hypermobility. This study delineates the clinical features associated with BRWD3 null variants and partial gene deletions, and suggests that BRWD3 should be included in the differential diagnosis of patients with an overgrowth-intellectual disability (OGID) phenotype, particularly in male patients with a mild or moderate intellectual disability associated with macrocephaly and/or obesity.

Antenatal diagnosis of cardio-facio-cutaneous syndrome: Prenatal characteristics and contribution of fetal facial dysmorphic signs in utero. About a case and review of literature.

Antenatal diagnosis of cardio-facio-cutaneous syndrome: prenatal characteristics and contribution of fetal facial dysmorphic signs in utero. This paper is a case study and review of literature. "RASopathies" is the term coined for a group of genetic diseases that share modulation inside the MAPKinase pathway. Mutations inside the coding sequence of any of these genes may be responsible for the upregulation of the RAS pathway, leading on the clinical level to Type 1 Neurofibromatosis (NF1), Noonan syndrome (NS), Costello syndrome (CS), Multiple Lentigines, Loose Anagen Hair syndrome, Cardio-Facio-Cutaneous syndrome (CFCS), and, more recently, Legius syndrome. While the postnatal presentation of this group is well-known, prenatal findings are less well recognized. The presence of a RASopathy during the prenatal period can be suspected on account of non-specific abnormalities: polyhydramnios, cystic hygroma or high nuchal translucency, macrosomia with proportionate short long bones, macrocephaly, renal, lymphatic, or cardiac defects. The current case report underlines the characteristic dysmorphic facial features on 3D-ultrasound (hypertelorism, down-slanting palpebral fissures, a long and marked philtrum, and low-set posteriorly rotated ears) that allow for a "RASopathy" to be postulated. After detecting a copy number variation (CNV) absence on a CGH array, we performed a RASopathy gene panel analysis, which identified a so-far unreported heterozygous de novo mutation in the BRAF gene (namely NM_004333.4 : c.1396 G > C ; p.Gly466Arg). Genetic counseling has, therefore, focused on the diagnosis of a RASopathy and predictable phenotype of CFCS, a distinct entity characterized by an increased risk of intellectual disability and early-onset feeding problems. We suggest that a more detailed prenatal facial evaluation should be performed in fetuses presenting high nuchal thickness, heart defects, or unusual findings, along with the absence of a CNV on a CGH array. Due to the dysmorphic facial features, targeted RASopathy genes are presumed to likely to be responsible for NS, CFCS, and CS.

Panel-Based Exome Sequencing for Neuromuscular Disorders as a Diagnostic Service.

BACKGROUND: Neuromuscular disorders (NMDs) are clinically and genetically heterogeneous. Accurate molecular genetic diagnosis can improve clinical management, provides appropriate genetic counseling and testing of relatives, and allows potential therapeutic trials. OBJECTIVE: To establish the clinical utility of panel-based whole exome sequencing (WES) in NMDs in a population with children and adults with various neuromuscular symptoms. METHODS: Clinical exome sequencing, followed by diagnostic interpretation of variants in genes associated with NMDs, was performed in a cohort of 396 patients suspected of having a genetic cause with a variable age of onset, neuromuscular phenotype, and inheritance pattern. Many had previously undergone targeted gene testing without results. RESULTS: Disease-causing variants were identified in 75/396 patients (19%), with variants in the three COL6-genes (COL6A1, COL6A2 and COL6A3) as the most common cause of the identified muscle disorder, followed by variants in the RYR1 gene. Together, these four genes account for almost 25% of cases in whom a definite genetic cause was identified. Furthermore, likely pathogenic variants and/or variants of uncertain significance were identified in 95 of the patients (24%), in whom functional and/or segregation analysis should be used to confirm or reject the pathogenicity. In 18% of the cases with a disease-causing variant of which we received additional clinical information, we identified a genetic cause in genes of which the associated phenotypes did not match that of the patients. Hence, the advantage of panel-based WES is its unbiased approach. CONCLUSION: Whole exome sequencing, followed by filtering for NMD genes, offers an unbiased approach for the genetic diagnostics of NMD patients. This approach could be used as a first-tier test in neuromuscular disorders with a high suspicion of a genetic cause. With uncertain results, functional testing and segregation analysis are needed to complete the evidence.

ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants.

PURPOSE: ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability. METHODS: By locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4. RESULTS: We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells. CONCLUSION: Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.

The Belgian MicroArray Prenatal (BEMAPRE) database: A systematic nationwide repository of fetal genomic aberrations.

OBJECTIVE: With the replacement of karyotyping by chromosomal microarray (CMA) in invasive prenatal diagnosis, new challenges have arisen. By building a national database, we standardize the classification and reporting of prenatally detected copy number variants (CNVs) across Belgian genetic centers. This database, which will link genetic and ultrasound findings with postnatal development, forms a unique resource to investigate the pathogenicity of variants of uncertain significance and to refine the phenotypic spectrum of pathogenic and susceptibility CNVs. METHODS: The Belgian MicroArray Prenatal (BEMAPRE) consortium is a collaboration of all genetic centers in Belgium. We collected data from all invasive prenatal procedures performed between May 2013 and July 2016. RESULTS: In this three-year period, 13 266 prenatal CMAs were performed. By national agreement, a limited number of susceptibility CNVs and no variants of uncertain significance were reported. Added values for using CMA versus conventional karyotyping were 1.8% in the general invasive population and 2.7% in cases with an ultrasound anomaly. Of the reported CNVs, 31.5% would have remained undetected with non-invasive prenatal test as the first-tier test. CONCLUSION: The establishment of a national database for prenatal CNV data allows for a uniform reporting policy and the investigation of the prenatal and postnatal genotype-phenotype correlation.

Familial Forms of Cushing Syndrome in Primary Pigmented Nodular Adrenocortical Disease Presenting with Short Stature and Insidious Symptoms: A Clinical Series.

Cushing syndrome (CS) is a rare disease in children, frequently associated with subtle or periodic symptoms that may delay its diagnosis. Weight gain and growth failure, the hallmarks of hypercortisolism in pediatrics, may be inconsistent, especially in ACTH-independent forms of CS. Primary pigmented nodular adrenocortical disease (PPNAD) is the rarest form of ACTH-independent CS, and can be associated with endocrine and nonendocrine tumors, forming the Carney complex (CNC). Recently, phenotype/genotype correlations have been described with particular forms of CNC where PPNAD is isolated or associated only with skin lesions. We present four familial series of CS due to isolated PPNAD, and compare them to available data from the literature. We discuss the clinical and molecular findings, and underline challenges in diagnosing PPNAD in childhood.

Patient with confirmed LEOPARD syndrome developing multiple melanoma.

LEOPARD syndrome, also known as Gorlin syndrome II, cardiocutaneous syndrome, lentiginosis profusa syndrome, Moynahan syndrome, was more recently coined as Noonan syndrome with multiple lentigines (NSML), inside the RASopathies. Historically, the acronym LEOPARD refers to the presence of distinctive clinical features such as: lentigines (L), electrocardiographic/conduction abnormalities (E), ocular hypertelorism (O), pulmonary stenosis (P), genital abnormalities (A), retardation of growth (R), and sensorineural deafness (D). This condition is identified in 85% of patients with phenotype hallmarks caused by presence a germline point mutation in PTPN11 gene. Association of melanoma to NSML seems to be rare: to our knowledge, two patients so far were reported in the literature. We herein present a patient diagnosed with LEOPARD syndrome, in whom molecular investigation confirmed the presence of the c.1403C>T mutation in exon 12 of the PTPN11 gene, who developed four superficial spreading melanomas and three atypical lentiginous hyperplasias. Three of the melanomas were achromic or hypochromic, three were in situ, and one had a Breslow index under 0.5 mm. Dermoscopic examination showed some characteristic white structures in most of the lesions, which were a signature pattern and a key for the diagnosis.