Noninvasive prenatal diagnosis of genetic diseases induced by triplet repeat expansion by linked read haplotyping and Bayesian approach.

The field of noninvasive prenatal diagnosis (NIPD) has undergone significant progress over the last decade. Direct haplotyping has been successfully applied for NIPD of few single-gene disorders. However, technical issues remain for triplet-repeat expansions. The objective of this study was to develop an NIPD approach for couples at risk of transmitting dynamic mutations. This method includes targeted enrichment for linked-read libraries and targeted maternal plasma DNA sequencing. We also developed an innovative Bayesian procedure to integrate the Hoobari fetal genotyping model for inferring the fetal haplotype and the targeted gene variant status. Our method of directly resolving parental haplotypes through targeted linked-read sequencing was smoothly performed using blood samples from families with Huntington's disease or myotonic dystrophy type 1. The Bayesian analysis of transmission of parental haplotypes allowed defining the genotype of five fetuses. The predicted variant status of four of these fetuses was in agreement with the invasive prenatal diagnosis findings. Conversely, no conclusive result was obtained for the NIPD of fragile X syndrome. Although improvements should be made to achieve clinically acceptable accuracy, our study shows that linked-read sequencing and parental haplotype phasing can be successfully used for NIPD of triplet-repeat expansion diseases.Trial registration: NCT04698551_date of first registration: 07/01/2021.

Pseudoxanthoma elasticum overlaps hereditary spastic paraplegia type 56.

PURPOSE: Pseudoxanthoma elasticum (PXE) is a recessive disorder involving skin, eyes and arteries, mainly caused by ABCC6 pathogenic variants. However, almost one fifth of patients remain genetically unsolved despite extensive genetic screening of ABCC6, as illustrated in a large French PXE series of 220 cases. We searched for new PXE gene(s) to solve the ABCC6-negative patients. METHODS: First, family-based exome sequencing was performed, in one ABCC6-negative PXE patient with additional neurological features, and her relatives. CYP2U1, involved in hereditary spastic paraplegia type 56 (SPG56), was selected based on this complex phenotype, and the presence of two candidate variants. Second, CYP2U1 sequencing was performed in a retrospective series of 46 additional ABCC6-negative PXE probands. Third, six additional SPG56 patients were evaluated for PXE skin and eye phenotype. Additionally, plasma pyrophosphate dosage and functional analyses were performed in some of these patients. RESULTS: 6.4% of ABCC6-negative PXE patients (n = 3) harboured biallelic pathogenic variants in CYP2U1. PXE skin lesions with histological confirmation, eye lesions including maculopathy or angioid streaks, and various neurological symptoms were present. CYP2U1 missense variants were confirmed to impair protein function. Plasma pyrophosphate levels were normal. Two SPG56 patients (33%) presented some phenotypic overlap with PXE. CONCLUSION: CYP2U1 pathogenic variants are found in unsolved PXE patients with neurological findings, including spastic paraplegia, expanding the SPG56 phenotype and highlighting its overlap with PXE. The pathophysiology of ABCC6 and CYP2U1 should be explored to explain their respective role and potential interaction in ectopic mineralization.

Severe Thoracic and Spinal Bone Abnormalities in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant, multi-system, neurocutaneous disorder that predisposes to the development of benign and malignant tumors. Classical skeletal abnormalities encompass sphenoid wing dysplasia, congenital bowing of the long bones and vertebral osteopathy associated with non-dystrophic or dystrophic scoliosis found in about 10% of NF1 patients. We report a 17-year-old boy affected by NF1 with extreme severe spinal and thoracic malformations affecting bone and lung tissues, including hypoplasia of the right lung, unilateral costal agenesis and severe dystrophic scoliosis characterized by association of hemivertebra, fusion of adjacent vertebral bodies and defective pedicles. At birth, he presented an acute respiratory distress requiring invasive ventilator support. The diagnosis of NF1 was confirmed at age 5 by the identification of a de novo heterozygous mutation c.4537C > T, p.Arg1513* in NF1. Trio-based Whole Exome Sequencing (WES) was performed to exclude coexistence of a second hit but no clearly other pathogenic variant has been identified. Until now, only one similar NF1 patient suffering from the same association of severe scoliosis and chest deformity leading to respiratory insufficiency was described. The severe prenatal NF1-related scoliosis could explain the lung abnormal development by absence of mechanical constraints. Severe Thoracic and Spinal Bone Abnormalities may be part of the NF1 bone phenotype and should be taken into account to allow adequate genetic counseling.

Implication of the SH3TC2 gene in Charcot-Marie-Tooth disease associated with deafness and/or scoliosis: Illustration with four new pathogenic variants.

The autosomal recessive demyelinating form of Charcot-Marie-Tooth can be due to SH3TC2 gene pathogenic variants (CMT4C, AR-CMTde-SH3TC2). We report on a series of 13 patients with AR-CMTde-SH3TC2 among a French cohort of 350 patients suffering from all type of inheritance peripheral neuropathy. The SH3TC2 gene appeared to be the most frequently mutated gene for demyelinating neuropathy in this series by NGS. Four new pathogenic variants have been identified: two nonsense variants (p.(Tyr970*), p.(Trp1199*)) and two missense variants (p.(Leu1126Pro), p.(Ala1206Asp)). The recurrent variant p.Arg954* was present in 62%, and seems to be a founder mutation. The phenotype is fairly homogeneous, as all these patients, except the youngest ones, presented scoliosis and/or hearing loss.

Truncating variants of the DLG4 gene are responsible for intellectual disability with marfanoid features.

Marfanoid habitus (MH) combined with intellectual disability (ID) is a genetically and clinically heterogeneous group of overlapping disorders. We performed exome sequencing in 33 trios and 31 single probands to identify novel genes specific to MH-ID. After the search for variants in known disease-causing genes and non-disease-causing genes with classical approaches, we searched for variants in non-disease-causing genes whose pLI was above 0.9 (ExAC Consortium data), in which truncating variants were found in at least 3 unrelated patients. Only DLG4 gene met these criteria. Data from the literature and various databases also indicated its implication in ID. DLG4 encodes post-synaptic density protein 95 (PSD-95), a protein expressed in various tissues, including the brain. In neurons, PSD-95 is located at the post-synaptic density, and is associated with glutamatergic receptor signaling (NMDA and AMPA). PSD-95 probably participates in dendritogenesis. Two patients were heterozygous for de novo frameshift variants and one patient carried a a consensus splice site variant. Gene expression studies supported their pathogenicity through haploinsufficiency and loss-of-function. Patients exhibited mild-to-moderate ID, similar marfanoid features, including a long face, high-arched palate, long and thin fingers, pectus excavatum, scoliosis and ophthalmological manifestations (nystagmus or strabismus). Our study emphasizes the role of DLG4 as a novel post-synaptic-associated gene involved in syndromic ID associated with MH.

Molecular, clinical and neuropsychological study in 31 patients with Kabuki syndrome and KMT2D mutations.

Kabuki syndrome (KS-OMIM 147920) is a rare developmental disease characterized by the association of multiple congenital anomalies and intellectual disability. This study aimed to investigate intellectual performance in children with KS and link the performance to several clinical features and molecular data. We recruited 31 children with KMT2D mutations who were 6 to 16 years old. They all completed the Weschler Intelligence Scale for Children, fourth edition. We calculated all indexes: the Full Scale Intellectual Quotient (FSIQ), Verbal Comprehension Index (VCI), Perceptive Reasoning Index (PRI), Processing Speed Index (PSI), and Working Memory Index (WMI). In addition, molecular data and several clinical symptoms were studied. FSIQ and VCI scores were 10 points lower for patients with a truncating mutation than other types of mutations. In addition, scores for FSIQ, VCI and PRI were lower for children with visual impairment than normal vision. We also identified a discrepancy in indexes characterized by high WMI and VCI and low PRI and PSI. We emphasize the importance of early identification and intensive care of visual disorders in patients with KS and recommend individual assessment of intellectual profile.

Further delineation of a rare recessive encephalomyopathy linked to mutations in GFER thanks to data sharing of whole exome sequencing data.

BACKGROUND: Alterations in GFER gene have been associated with progressive mitochondrial myopathy, congenital cataracts, hearing loss, developmental delay, lactic acidosis and respiratory chain deficiency in 3 siblings born to consanguineous Moroccan parents by homozygosity mapping and candidate gene approach (OMIM#613076). Next generation sequencing recently confirmed this association by the finding of compound heterozygous variants in 19-year-old girl with a strikingly similar phenotype, but this ultra-rare entity remains however unknown from most of the scientific community. MATERIALS AND METHODS: Whole exome sequencing was performed as part of a "diagnostic odyssey" for suspected mitochondrial condition in 2 patients, presenting congenital cataracts, progressive encephalomyopathy and hypotrophy and detected unreported compound heterozygous variants in GFER. RESULTS: Thanks to an international data sharing, we found 2 additional patients carrying compound heterozygous variants in GFER. Reverse phenotyping confirmed the phenotypical similarities between the 4 patients. Together with the first literature reports, the review of these 8 cases from 4 unrelated families enables us to better describe this apparently homogeneous disorder, with the clinical and biological stigmata of mitochondrial disease. CONCLUSION: This report highlights the clinical utility of whole exome sequencing and reverse phenotyping for the diagnosis of ultra-rare diseases and underlines the importance of a broad data sharing for accurate clinical delineation of previously unrecognized entities.

[Acid sphingomyelinase deficiency (Niemann-Pick disease type B) in adulthood: A retrospective multicentric study of 28 adult cases]. / Déficit en sphingomyélinase acide (maladie de Niemann-Pick B) : une étude rétrospective multicentrique de 28 patients adultes.

INTRODUCTION: Acid sphingomyelinase deficiency (ASMD) is an autosomal recessive disease with a clinical spectrum ranging from a neurovisceral infantile form (Niemann-Pick disease type A) to a chronic visceral form also encountered in adults (Niemann-Pick disease type B, NP-B). METHODS: Retrospective multicentric analysis of French adult patients with ASMD over the period 1985-March 2015. Clinical, biological, and imaging data were analyzed. RESULTS: Twenty-eight patients (19 males, 9 females) were analyzed. Diagnosis was made before the age of 10 years in 16 cases. Main symptoms at diagnosis were spleen/liver enlargement and interstitial lung disease. Biological abnormalities included: thrombocytopenia (platelet count <150 000/mm3) in 24 cases including 4 patients with platelet count <60 000/mm3, constantly low high-density lipoprotein (HDL) cholesterol, polyclonal hypergammaglobulinemia (n=6), monoclonal gammopathy of unknown significance (n=5), normal prothrombin level discordant with low factor V (n=5), elevated chitotriosidase level (n=11). The diagnosis was confirmed in all cases by deficient acid sphingomyelinase enzyme activity. SMPD1 gene sequencing was performed in 25 cases. The frequent p.R610del mutation was largely predominant, constituting 62% of the non-related alleles. During the follow-up period, three patients died before 50 years of age from cirrhosis, heart failure and lung insufficiency, respectively. CONCLUSION: ASMD in adulthood (NP-B) associates spleen/liver enlargement and interstitial lung disease. Early diagnosis and appropriate management are essential for reducing the risk of complications, improving quality of life, and avoiding inappropriate procedures such as splenectomy. To date, only symptomatic therapy is available. A phase 2/3 therapeutic trial with IV infusion of recombinant enzyme is on-going.

Classifications of neurogenetic diseases: An increasingly complex problem.

Neurodegenerative disorders represent a wide group of diseases affecting the central and/or peripheral nervous system. Many of these disorders were described in the 19th century, but our genetic knowledge of them is recent (over the past 25 years). However, the continual discovery of disease-causing gene mutations has led to difficulties in the classification of these diseases. For this reason, our present proposals for updating and simplifying the classification of some of these conditions (Charcot-Marie-Tooth diseases, distal hereditary motor neuropathies, hereditary sensory and autonomic neuropathies, hereditary spastic ataxias, hereditary spastic paraplegias and hereditary spastic ataxias) are expounded here.

Fetal phenotypes in otopalatodigital spectrum disorders.

Otopalatodigital spectrum disorders (OPDSD) include OPD syndromes types 1 and type 2 (OPD1, OPD2), Melnick-Needles syndrome (MNS), and frontometaphyseal dysplasia (FMD). These conditions are clinically characterized by variable skeletal dysplasia associated in males, with extra-skeletal features including brain malformations, cleft palate, cardiac anomalies, omphalocele and obstructive uropathy. Mutations in the FLNA gene have been reported in most FMD and OPD2 cases and in all instances of typical OPD1 and MNS. Here, we report a series of 10 fetuses and a neonatally deceased newborn displaying a multiple congenital anomalies syndrome suggestive of OPDSD and in whom we performed FLNA analysis. We found a global mutation rate of 44%. This series allows expanding the clinical and FLNA mutational spectrum in OPDSD. However, we emphasize difficulties to correctly discriminate OPDSD based on clinical criteria in fetuses due to the major overlap between these conditions. Molecular analyses may help pathologists to refine clinical diagnosis according to the type and the location of FLNA mutations. Discriminating the type of OPDSD is of importance in order to improve the genetic counseling to provide to families.

RECURRENCE OF POMPE DISEASE IN FIRST COUSINS.

We report on the cases of two first-degree non-consanguineous cousins with infantile-onset Pompe disease, a rare autosomal recessive disease. The first patient developed cardiorespiratory failure at age 1 year. When she was 4 her male cousin developed hypotonia during his first month of life. Both infants had cardiac hypertrophy at diagnosis and shared the c.1927G>A missense mutation. Since a first degree cousin of an affected patient has 50 times the risk of developing the disease compared with unrelated infants and since cardiac hypertrophy is constant in affected infants, the combination of cardiac symptoms with a history of Pompe disease in a first degree cousin leads to a very high probability of having the condition. Clinically oriented screening based on simple diagnostic procedures such as echocardiogram and anamnesis could accelerate the initiation of enzyme replacement therapy of the deficient acid α-glucosidase which is critical to restoring cardiac function in affected infants.

[Significance of the urine strip test in case of stunted growth]. / Intérêt de l'examen des urines à la bandelette en cas de retard de croissance.

Observation of stunted growth in children usually leads the general practitioner to refer the patient to endocrinologists or gastroenterologists. In most cases, after a complementary check-up, the diagnosis is made and treatment is initiated. However, certain cases remain undiagnosed, particularly renal etiologies, such as proximal tubulopathy. The urine strip test at the initial check-up would be an easy and inexpensive test to avoid delayed diagnosis. The aim of the present paper is to increase general physicians' and pediatricians' awareness of the significance of questioning the parents and using the urine strip test for any child presenting stunted growth. We report a patient case of a 20-month-old child admitted to the emergency department for severe dehydration. He had displayed stunted growth since the age of 5 months and showed a negative etiologic check-up at 9 months of age. Clinical examination at admission confirmed stunted growth with loss of 2 standard deviations and signs of dehydration with persistent diuresis. Skin paleness, ash-blond hair, and signs of rickets were also observed and the urine strip test showed positive pads for glycosuria and proteinuria. Polyuria and polydipsia were also revealed following parents' questioning, suggesting proximal tubulopathy (Fanconi syndrome). Association of stunted growth, rickets, polyuria and polydipsia, glycosuria (without ketonuria and normal glycemia), and proteinuria suggest nephropathic cystinosis. Ophthalmic examination showed cystine deposits in the cornea. The semiotic diagnosis of nephropathic cystinosis was confirmed by leukocyte cystine concentrations and genetic investigations. This case report clearly illustrates the significance of the urine strip test to easily and quickly concentrate the diagnosis of stunted growth on a renal etiology (glycosuria, proteinuria), especially on proximal tubulopathy for which the most frequent cause is nephropathic cystinosis. Specificity of nephropathic cystinosis treatment is that the age of treatment initiation is crucial and determinant for the prognosis of the disease and the onset of final stage renal failure. Therefore, the urine strip test should be included in the systematic check-up of stunted growth to identify any renal etiology.

[Cavitary lesions in multiple sclerosis: multicenter study on twenty patients]. / Formes cavitaires de sclérose en plaques: étude multicentrique sur vingt patients.

INTRODUCTION: Cavitary white matter changes are mainly described in leukodystrophies and especially in vanishing white matter disease. Large cavitary lesions are not typical for multiple sclerosis (MS). METHODS: We studied MS patients with large cavitary brain lesions. Patient characteristics, disease onset/duration/subtype, expanded disability status scale (EDSS), mini mental state (MMS), vanishing white matter disease genetic analysis, and MRI characteristics of the cavitary lesions were analyzed. RESULTS: Twenty patients were analyzed (6 men and 14 women). Mean age at disease onset was 37.6 (range 17-58). Mean disease duration was 10 years (range 2-20). Five patients had initial relapsing-remitting MS and nine patients had primary-progressive MS. Mean EDSS was 5.5 (range 2-8). Mean MMS was 20/30. Vanishing white matter disease genetic analysis was performed and negative in seven patients. Inferior corpus callosum lesions were seen in all patients with available sagittal FLAIR sequences. Cavitary lesions were strictly supratentorial, and located inside the diffuse leukoencephalopathy, with often a posterior predominance. CONCLUSION: MS patients with large cavitary lesions seem to represent a MS subgroup, predominantly women, with relatively late disease onset, predominantly primary-progressive type, relatively high EDSS scores, and severe cognitive dysfunction.

Systematic molecular and cytogenetic screening of 100 patients with marfanoid syndromes and intellectual disability.

The association of marfanoid habitus (MH) and intellectual disability (ID) has been reported in the literature, with overlapping presentations and genetic heterogeneity. A hundred patients (71 males and 29 females) with a MH and ID were recruited. Custom-designed 244K array-CGH (Agilent®; Agilent Technologies Inc., Santa Clara, CA) and MED12, ZDHHC9, UPF3B, FBN1, TGFBR1 and TGFBR2 sequencing analyses were performed. Eighty patients could be classified as isolated MH and ID: 12 chromosomal imbalances, 1 FBN1 mutation and 1 possibly pathogenic MED12 mutation were found (17%). Twenty patients could be classified as ID with other extra-skeletal features of the Marfan syndrome (MFS) spectrum: 4 pathogenic FBN1 mutations and 4 chromosomal imbalances were found (2 patients with both FBN1 mutation and chromosomal rearrangement) (29%). These results suggest either that there are more loci with genes yet to be discovered or that MH can also be a relatively non-specific feature of patients with ID. The search for aortic complications is mandatory even if MH is associated with ID since FBN1 mutations or rearrangements were found in some patients. The excess of males is in favour of the involvement of other X-linked genes. Although it was impossible to make a diagnosis in 80% of patients, these results will improve genetic counselling in families.

Sight-threatening phenylketonuric encephalopathy in a young adult, reversed by diet.

Phenylketonuria (PKU) leads to severe neurological disorders in childhood, shunned by the diet. The long-term prognosis after diet diversification at adolescence is uncertain. We report a case of cortical blindness in a young patient regressive 1 month after the diet was resumed.Mr M., 25 years old, had PKU detected at birth. He maintained good serum levels of Phenylalanine (Phe) (120-300 µmol/L) during childhood and got a normal intellectual development. During adolescence he diversified his diet but maintained low meat and fish intake; Phe was ~1,200 µmol/L with no symptoms. In 2009, the patient stopped the low-Phe amino acid substitutes due to weariness. On June 27, 2011, he consulted for a decrease of visual acuity progressing for 6 months. Ophthalmologic examination found that visual acuity was 2/10 in two eyes associated to a central visual field defect. The visual evoked potentials were altered. MRI showed bilateral and symmetric occipital FLAIR hyperintensities. On admission in the Nutrional Unit on June 29, 2011, blood pressure was 120/70 mmHg, there was no other neurological abnormality. Phe was at 1,512 µmol/L, and not responsive to BH4. He was then treated with a very low-Phe diet with an amino acid substitute, and he obtained Phe between 120 and 300 µmol/L. Visual acuity was suddenly restored on August 1, 2011, with a dramatic attenuation of the MRI hyperintensities.Our observation shows that the withdrawal of the diet and substitutes exposes to serious neurological complications in adults that may reverse with a fast nutritional support.

[Natural history of adult-onset eIF2B-related disorders: a multicentric survey of 24 cases]. / Histoire naturelle des leucodystrophies avec mutation EIF2B: étude rétrospective multicentrique de 24 cas adultes.

INTRODUCTION: The childhood ataxia with central nervous system hypomyelination-vanishing white matter syndrome (CACH-VWM) was first characterized in children (2-5 years) on clinical and MRI criteria: cerebellospastic signs associated with episodes of rapid deterioration following stress and extensive cavitatingleucoencephalopathy. Causative mutations were found in the five genes encoding the subunits of the eukaryotic initiation factor 2B (eIF2B), involved in protein synthesis and its regulation under cellular stresses. A broad clinical spectrum has been subsequently described from congenital to adult-onset forms leading to the concept of eIF2B-related disorders. Our aim was to describe clinical and brain magnetic resonance imaging characteristics, genetic findings and natural history of patients with adult-onset eIF2B-related disorders. METHODS: The inclusion criteria were based on the presence of EIF2B mutations and a disease onset after the age of 16 years. One patient with an asymptomatic diagnosis was also included. Clinical and MRI findings were retrospectively recorded in all patients. This multicentric study included 24 patients from 22 families. RESULTS: A sex-ratio imbalance was noted (male/female=5/19). The mean age of onset was 30 years (range 12-62). Initial symptoms were neurologic (n=20), psychiatric (n=3) and ovarian failure (n=6). During follow-up (mean: 11 years, range 2-35 years), two patients died. Of the 22 survivors, 67% showed a decline in their cognitive functions and mean EDSS was 5.6 (range=0-9.5). One case remained asymptomatic. Stress worsened clinical symptoms in 33% of the patients. Magnetic resonance imaging findings consisted of cerebral atrophy (92%), extensive cystic leucoencephalopathy (83%), corpus callosum involvement (92%) and cerebellar (37%) T2-weighted hyperintensities. Most patients (83%) showed mutations in the EIF2B5 gene. The recurrent p.Arg113His-eIF2Be mutation was found at a homozygous state in 58% of the 24 eIF2B-mutated patients. CONCLUSION: eIF2B-related disorder is probably underestimated as an adult-onset inherited leucoencephalopathy. Cerebral atrophy is constant, whereas the typical vanishing of the white matter can be absent. Functional and cognitive prognosis remains severe. Molecular diagnosis is facilitated for these forms by screening for the recurrent p.Arg113His-eIF2Be mutation.

Oculo-dento-digital dysplasia: lack of genotype-phenotype correlation for GJA1 mutations and usefulness of neuro-imaging.

Oculo-dento-digital dysplasia (ODDD) is an autosomal dominant disorder with complete penetrance and high intra- and interfamilial phenotypic variability. The key features in this syndrome are microphthalmia, enamel hypoplasia and syndactyly of the 4th-5th fingers. ODDD is caused by mutations in the connexin 43 gene (GJA1). We report here four patients from three families with GJA1 mutations, one of them diagnosed prenatally. The three mutations (c.52T > C/p.Ser18Pro, c.689_690delTA/p.Tyr230CysfsX6, c.442C > G/p.Arg148Gly) have been reported once before. Two patients had white matter hypersignal anomalies, associated in one case with mental retardation, but asymptomatic in the other one, an observation that leads us to discuss systematic neuroradiological imaging for ODDD. One case has optic atrophy, another has hypospadias. The patient carrying a truncating mutation of Cx43 did not have palmoplantar keratoderma, in contradiction with the previously suggested genotype-phenotype correlation between truncating mutation and skin involvement.

Bilateral periventricular nodular heterotopia in France: frequency of mutations in FLNA, phenotypic heterogeneity and spectrum of mutations.

Bilateral periventricular nodular heterotopia (BPNH) is the most common form of periventricular heterotopia. Mutations in FLNA, encoding filamin A, are responsible for the X linked dominant form of BPNH (FLNA-BPNH). Recently, atypical phenotypes including BPNH with Ehlers-Danlos syndrome (BPNH-EDS) have been recognised. A total of 44 FLNA mutations have so far been reported in this phenotype. Most of these mutations lead to a truncated protein, but few missense mutations have also been described. Here, the results of a mutation screening conducted in a series of 32 BPNH patients with the identification of 12 novel point mutations in 15 patients are reported. Nine mutations were truncating, while three were missense. Three additional patients with BPNH-EDS and a mutation in FLNA are described. No phenotype-genotype correlations could be established, but these clinical data sustain the importance of cardiovascular monitoring in FLNA-BPNH patients.