[Newborn screening in France: news and perspectives]. / Le dépistage néonatal en France : actualités et perspectives.

Newborn screening is a major public health concern. In France, it was established in 1972 with systematic screening for phenylketonuria. Subsequently, other screenings, including congenital hypothyroidism, congenital adrenal hyperplasia, cystic fibrosis, and sickle cell disease, were added. The introduction of tandem mass spectrometry in screening laboratories in 2020 enabled the inclusion of eight additional inherited metabolic diseases: aminoacidopathies (tyrosinemia type I, maple syrup urine disease, and homocystinuria), organic acidurias (isovaleric and glutaric type I acidurias), and disorders of fatty acid metabolism (MCADD, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), and primary carnitine deficiency). We briefly present these newly added diseases, of which public awareness is still incomplete.

Wagner syndrome: Novel VCAN variant and prophylactic management with encircling band and retinopexy.

Purpose: Wagner syndrome is an autosomal genetic vitreoretinopathy characterized by chorioretinal atrophy, avascular vitreous veils, reduced visual acuity and early retinal detachment in advanced cases. Management of Wagner syndrome usually results in observation then management of occurring complications. Observations: We report the case of a 9-year-old girl presenting with supposed Wagner syndrome that we managed with prophylactic encircling band and retinopexy in both eyes. The genetic testing revealed a new variant in the intron 7 non canonical splice acceptor site, c.4004-12_4004-6delins17, that was also present in her father. Conclusions and Importance: The VCAN variant found in this proband and her father has not been described yet but shows high predictions of pathogenicity. The previous reported variants in VCAN intron 7 and the associated phenotype for both cases allowed us to attribute this variant to Wagner syndrome. In Wagner syndrome, management is usually curative. After prophylactic surgery in our case, the zones of retinal delamination were well supported by the scleral buckle, releasing the vitreoretinal tractions, and the additional laser focalized on the temporal zones of dehiscence secured the retina. An encircling band may be a good way to prevent RD in patients with Wagner syndrome at risk.

Stargardt disease-associated in-frame ABCA4 exon 17 skipping results in significant ABCA4 function.

BACKGROUND: ABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping. METHODS: A STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids. RESULTS: Late onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids. CONCLUSIONS: Exon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients.

Systematic thyroid screening in myotonic dystrophy: link between thyroid volume and insulin resistance.

BACKGROUND: Myotonic dystrophy (DM1), a neuromuscular disease related to DMPK gene mutations, is associated to endocrine disorders and cancer. A routine endocrine work-up, including thyroid ultrasound (US), was conducted in 115 genetically-proven DM1 patients in a neuromuscular reference center. The aim of this study was to determine the prevalence and the causes of US thyroid abnormalities in DM1. RESULTS: In the whole population (age 45.1 ± 12.2 years, 61.7% female), palpable nodules or goiters were present in 29.2%. The percentage of US goiter (thyroid volume > 18 mL) and US nodules were, respectively, 38.3 and 60.9%. Sixteen of the 115 patients had a thyroidectomy, after 22 fine-needle aspiration cytology guided by thyroid imaging reporting and data system (TIRADS) classification. Six micro- (1/6 pT3) and 3 macro-papillary thyroid carcinoma (PTCs) (2/3 intermediate risk) were diagnosed (7.9% of 115). Thyroid US led to the diagnosis of 4 multifocal and 2 unifocal (including 1 macro-PTC) non-palpable PTCs. Ultrasound thyroid volume was positively correlated to body mass index (BMI) (p = 0.015) and parity (p = 0.036), and was inversely correlated to TSH (p < 0.001) and vitamin D levels (p = 0.023). The BMI, the frequencies of glucose intolerance and PTC were significantly higher in UsGoiter versus non-UsGoiter groups. CONCLUSION: In this systematically screened DM1 cohort, the frequency of UsGoiter, mainly associated to BMI, was about 40%, US nodules 60%, thyroidectomies 13-14%, and PTCs 8%, two-thirds of them being micro-PTCs with good prognosis. Therefore, a systematic screening remains debatable. A targeted US screening in case of clinical abnormality or high BMI seems more appropriate.

Phenotypic Characteristics of a French Cohort of Patients with X-Linked Retinoschisis.

PURPOSE: To analyze the retinal structure in patients with X-linked retinoschisis (XLRS) using spectral-domain OCT and to correlate the morphologic findings with visual acuity, electroretinographic results, and patient age. DESIGN: Retrospective, observational study. PARTICIPANTS: Data from 52 consecutive male patients with molecularly confirmed XLRS were collected retrospectively. METHODS: Complete clinical evaluation included best-corrected visual acuity, full-field electroretinography, fundus photography, spectral-domain OCT, and fundus autofluorescence. Spectral-domain OCT images were analyzed to determine full thickness of the retina and tomographic structural changes. MAIN OUTCOME MEASURES: Relationships between age, OCT, and visual acuity were assessed. RESULTS: One hundred four eyes of 52 patients were included. The mean age at inclusion was 24±15 years (range, 3-57 years). The best-corrected visual acuity ranged from no light perception to 0.1 logarithm of the minimum angle of resolution (mean, 0.6±0.38 logarithm of the minimum angle of resolution). Macular schisis was found in 88% of eyes and macular atrophy was found in 11% of eyes, whereas peripheral schisis was present in 30% of eyes. A spoke-wheel pattern of high and low intensity was the most frequently observed fundus autofluorescence abnormality (51/94 eyes [54%]). The b-to-a amplitude ratio on bright-flash dark-adapted electroretinography was reduced significantly in 45 of 64 eyes (70%). Spectral-domain OCT was available for 97 eyes and showed foveoschisis in 76 of 97 eyes (78%), parafoveal schisis in 10 of 97 eyes (10%), and foveal atrophy in 11 of 97 eyes (11%). Mean central macular thickness (CMT) was of 373.6±140 µm. Cystoid changes were localized mainly in the inner nuclear layer (85/97 eyes [88%]). Qualitative defects in photoreceptor structures were found in most eyes (79/97 eyes [81%]), and the most frequent abnormality was an interruption of the photoreceptor cell outer segment tips (79/79 eyes [100%]). Older age correlated well with lower CMT (correlation coefficient [CC], -0.44; P < 0.001) and with lower photoreceptor outer segment (PROS) length (CC, -0.42; P < 0.001). Lower visual acuity correlated strongly with lower PROS length (CC, -0.53; P < 0.001). CONCLUSIONS: This study underlined the wide variety of clinical features of XLRS. It highlighted the correlation between visual acuity, patient age, and OCT features, emphasizing the relevance of the latter as potential outcome measure in clinical trials.

A new autosomal dominant eye and lung syndrome linked to mutations in TIMP3 gene.

To revisit the autosomal dominant Sorsby fundus dystrophy (SFD) as a syndromic condition including late-onset pulmonary disease. We report clinical and imaging data of ten affected individuals from 2 unrelated families with SFD and carrying heterozygous TIMP3 mutations (c.572A > G, p.Y191C, exon 5, in family 1 and c.113C > G, p.S38C, exon 1, in family 2). In family 1, all SFD patients older than 50 (two generations) had also a severe emphysema, despite no history of smoking or asthma. In the preceding generation, the mother died of pulmonary emphysema and she was blind after the age of 50. Her two great-grandsons (<20 years), had abnormal Bruch Membrane thickness, a sign of eye disease. In family 2, eye and lung diseases were also associated in two generations, both occurred later, and lung disease was moderate (bronchiectasis). This is the first report of a syndromic SFD in line with the mouse model uncovering the role of TIMP3 in human lung morphogenesis and functions. The TIMP3 gene should be screened in familial pulmonary diseases with bronchiectasis, associated with a medical history of visual loss. In addition, SFD patients should be advised to avoid tobacco consumption, to practice sports, and to undergo regular pulmonary examinations.

Distinctive Patterns of Transthyretin Amyloid in Salivary Tissue: A Clinicopathologic Study of 92 Patients With Amyloid-containing Minor Salivary Gland Biopsies.

Accurate typing of amyloidosis is still a major issue for pathologists and clinicians. Besides clinical data and immunohistochemistry, the histologic distribution of amyloid could represent a useful tool to prevent typing errors, such as the misdiagnosis of hereditary and senile amyloidosis as light chain-related amyloidosis (AL). Minor salivary gland biopsy (MSGB) is a widely performed procedure for amyloidosis diagnosis and typing. In the largest clinicopathologic series of amyloid-containing MSGB specimens to date, we investigated for the first time whether amyloidosis subtypes can be distinguished according to their pattern of salivary amyloid deposition. The histologic distribution and semiquantification of amyloid within salivary tissue were thoroughly reassessed for each case using Congo red-fluorescence. Clinical data were retrospectively collected. The cohort included 92 patients with amyloid-containing minor salivary gland biopsies. The type of amyloidosis was AL in 51 patients (55.4%), non-V30M mutant ATTR in 10 (10.9%), V30M mutant ATTR in 8 (8.7%), serum amyloid A-derived amyloidosis (AA) in 6 (6.5%), wild-type ATTR in 4 (4.3%), gelsolin in 3 (3.3%), and unclassified in 10 (10.9%). Amyloid was more abundant in AL and AA compared with ATTR amyloidosis, because of more extensive basement membranes and vascular deposits. Conversely, non-V30M mutant ATTR and wt-ATTR were strongly associated with peculiar amyloid nodules located in close contact with salivary excretory ducts, with a specificity of 91.7%. In conclusion, our study suggests for the first time that MSGB, in addition to its high sensitivity for amyloidosis diagnosis, is a simple and effective tool for the recognition of ATTR amyloidosis.

Relative frequencies of inherited retinal dystrophies and optic neuropathies in Southern France: assessment of 21-year data management.

PURPOSE: Inherited retinal dystrophies (IRDs) and inherited optic neuropathies (IONs) are rare diseases defined by specific clinical and molecular features. The relative prevalence of these conditions was determined in Southern France. METHODS: Patients recruited from a specialized outpatient clinic over a 21-year period underwent extensive clinical investigations and 107 genes were screened by polymerase chain reaction/sequencing. RESULTS: There were 1957 IRD cases (1481 families) distributed in 70% of pigmentary retinopathy cases (56% non-syndromic, 14% syndromic), 20% maculopathies and 7% stationary conditions. Patients with retinitis pigmentosa were the most frequent (47%) followed by Usher syndrome (10.8%). Among non-syndromic pigmentary retinopathy patients, 84% had rod-cone dystrophy, 8% cone-rod dystrophy and 5% Leber congenital amaurosis. Macular dystrophies were encountered in 398 cases (30% had Stargardt disease and 11% had Best disease). There were 184 ION cases (127 families) distributed in 51% with dominant optic neuropathies, 33% with recessive/sporadic forms and 16% with Leber hereditary optic neuropathy. Positive molecular results were obtained in 417/609 families with IRDs (68.5%) and in 27/58 with IONs (46.5%). The sequencing of 5 genes (ABCA4, USH2A, MYO7A, RPGR and PRPH2) provided a positive molecular result in 48% of 417 families with IRDs. Except for autosomal retinitis pigmentosa, in which less than half the families had positive molecular results, about 75% of families with other forms of retinal conditions had a positive molecular diagnosis. CONCLUSIONS: Although gene discovery considerably improved molecular diagnosis in many subgroups of IRDs and IONs, retinitis pigmentosa, accounting for almost half of IRDs, remains only partly molecularly defined.

Renal involvement in MELAS syndrome - a series of 5 cases and review of the literature.

Renal dysfunction is increasingly recognized as a potential clinical feature of mitochondrial cytopathies such as mitochondrial encephalomyopathy, lacticacidosis and stroke-like episodes (MELAS) syndrome. Five cases of MELAS syndrome with renal involvement from 4 unrelated families are presented in this case series. Three of the 5 patients had a history of maternally-inherited diabetes and/or deafness. Focal and segmental glomerulosclerosis and arteriolar hyaline thickening were the most striking findings on renal biopsy. In addition to clinical presentation with the typical symptoms of MELAS syndrome, genetic testing in these patients identified the A3243G point mutation in the tRNALeu gene of the mitochondrial DNA (mtDNA). The diagnosis of MELAS syndrome was thus considered to be unequivocal. The incidence of kidney disease in MELAS syndrome may be underestimated although a study is required to investigate this hypothesis. As the A3243G mtDNA mutation leads to a progressive adult-onset form of focal segmental glomerulosclerosis (FSGS), screening for the MELAS A3243G mtDNA mutation should therefore be performed especially in patients with maternally-inherited diabetes or hearing loss presenting with FSGS.

Transcriptional regulation of the human ST6GAL2 gene in cerebral cortex and neuronal cells.

The second human beta-galactoside alpha-2,6-sialyltransferase (hST6Gal II) differs from hST6Gal I, the first member of ST6Gal family, in substrate specificity and tissue expression pattern. While ST6GAL1 gene is expressed in almost all human tissues, ST6GAL2 shows a restricted tissue-specific pattern of expression, mostly expressed in embryonic and adult brain. In order to understand the mechanisms involved in the transcriptional regulation of ST6GAL2, we first characterized the transcription start sites (TSS) in SH-SY5Y neuroblastoma cells. 5' RACE experiments revealed multiple TSS located on three first alternative 5' exons, termed EX, EY and EZ, which are unusually close on the genomic sequence and are all located more than 42 kbp upstream of the first common coding exon. Using Taqman duplex Q-PCR, we showed that the ST6GAL2 transcripts initiated by EX or EY are mainly expressed in both brain-related cell lines and human cerebral cortex, testifying for the use of a similar transcriptional regulation in vivo. Furthermore, we also showed for the first time hST6Gal II protein expression in the different lobes of the human cortex. Luciferase reporter assays allowed us to define two sequences upstream EX and EY with a high and moderate promoter activity, respectively. Bioinformatics analysis and site-directed mutagenesis showed that NF-kappaB and NRSF are likely to act as transcriptional repressors, whereas neuronal-related development factors Sox5, Puralpha and Olf1, are likely to act as transcriptional activators of ST6GAL2. This suggests that ST6GAL2 transcription could be potentially activated for specific neuronal functions.

A genetic variation in the ADORA2A gene modifies age at onset in Huntington's disease.

Based on the pathophysiological role of adenosine A(2A) receptors in HD, we have evaluated the association of the 1976C/T single-nucleotide polymorphism in the ADORA2A gene (rs5751876) with residual age at onset (AAO) in HD. The study population consisted of 791 unrelated patients belonging to the Huntington French Speaking Network. The variability in AAO attributable to the CAG repeats number was calculated by linear regression using the log (AAO) as the dependent variable, and the respective rs5751876 genotypes as independent variables. We show that the rs5751876 variant significantly influences the variability in AAO. The R(2) statistic rose slightly but significantly (p=0.019) when rs5751876 T/T genotype was added to the regression model. Patients harbouring T/T genotype have an earlier AAO of 3.8 years as compared to C/C genotype (p=0.02). Our data thus strengthens the pathophysiological role of A(2A) receptors in Huntington's disease.

Altered splicing of Tau in DM1 is different from the foetal splicing process.

Among the different mechanisms underlying the etiopathogenesis of myotonic dystrophy type 1 (DM1), a backward reprogramming to a foetal splicing machinery is an interesting hypothesis. To address this possibility, Tau splicing, which is regulated during development and modified in DM1, was analyzed. Indeed, a preferential expression of the foetal Tau isoform, instead of the six normally found, is observed in adult DM1 brains. By using two cell lines, we show here that the cis-regulating elements necessary to generate the unique foetal Tau isoform are dispensable to reproduce the trans-dominant effect induced by DM1 mutation on Tau exon 2 inclusion. Our results suggest that the mis-splicing of Tau in DM1 is resulting from a disease-associated mechanism.

ETR-3 represses Tau exons 2/3 inclusion, a splicing event abnormally enhanced in myotonic dystrophy type I.

Altered splicing of transcripts, including the insulin receptor (IR) and the cardiac troponin (cTNT), is a key feature of myotonic dystrophy type I (DM1). CELF and MBNL splicing factor members regulate the splicing of those transcripts. We have previously described an alteration of Tau exon 2 splicing in DM1 brain, resulting in the favored exclusion of exon 2. However, the factors required for alternative splicing of Tau exon 2 remain undetermined. Here we report a decreased expression of CELF family member and MBNL transcripts in DM1 brains as assessed by RT-PCR. By using cellular models with a control- or DM1-like splicing pattern of Tau transcripts, we demonstrate that ETR-3 promotes selectively the exclusion of Tau exon 2. These results together with the analysis of Tau exon 6 and IR exon 11 splicing in brain, muscle, and cell models suggest that DM1 splicing alteration of several transcripts involves various factors.