Endophilin A2 Deficiency Impairs Antibody Production in Humans.

Endophilin A2, the sole endophilin A family member expressed in hematopoietic cells, regulates various aspects of membrane dynamics, including autophagy and endocytosis. Recent studies in rodents highlight the essential role of endophilin A2 in modulating immune responses. Here we report a homozygous frameshift variant in the SH3GL1 gene (NM_003025.3:c.427delC; p.Leu143Serfs*9), detected by whole exome sequencing in a 14-year-old boy with predominantly antibody deficiency. The patient who is issued from a consanguineous Lebanese family, presents since the age of 18 months with recurrent respiratory tract infections, low peripheral B cell counts and pan-hypogammaglobulinemia, with no history of opportunistic infections. This defect is associated with decrease in switched memory B cells development, impaired in-vitro B cell proliferation and diminished in-vitro IgG production. The detected variant in SH3GL1 segregates with the disease in the family. It significantly decreases the expression of the protein in the patient's peripheral blood compared to healthy controls, thus confirming its pathogenicity. Interestingly, endophilin A2-deficient Sh3gl1-/- mice have been reported to present defects in germinal center B cell responses and in the production of high-affinity IgG. Our data suggests that endophilin A2 deficiency impairs antibody production in humans. Reporting further cases with mutations in SH3GL1 is needed to better characterize the inborn error of immunity linked to this gene.

Expanding the phenotypes of ABL1 deficiency syndromes: When mutations in different isoforms Lead to different diseases.

All reported ABL1 gain of function and loss of function (LOF) variants, impact both isoforms 1a and 1b. Our findings suggest that LOF variants affecting solely ABL1 isoform 1b may lead to a distinct autosomal recessive new phenotype.

Three Complex alleles associated with N1303K mutation and their molecular consequences.

Cystic Fibrosis (CF) in Arab Mediterranean countries has a different CFTR mutational profile if compared either to Caucasians or in the Arabian Peninsula. The c.3909C>G (N1303K, p.Asn1303Lys) mutation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). This mutation represents a higher frequency in the Mediterranean countries in association with different polymorphisms or mutations in cis position constituting various complex alleles. N1303K mutation induces many phenotypes, especially pancreatic insufficiency from mild to severe and it is associated in cis with other polymorphisms. The aim of this investigation is therefore to screen complex alleles carrying N1303K mutation among Lebanese, Egyptian and French patients. All exons of the CFTR and their flanking regions were performed by PCR amplification, followed by automated direct DNA sequencing. Two complex alleles are more frequent corresponding to Wild Type and mutated haplotype. Besides that two other very rare complex alleles have been detected, one in Egyptian and French samples, and then another one in Lebanon samples. We have studied their impact on the CFTR mRNA splicing using a minigene strategy. Constructs containing wild-type and mutant CFTR cloned into the pTBNdeI hybride minigene have been expressed in HeLa, HT29 and HEK293 cells. RT-PCR analysis of mRNA using ß-globin-specific primers revealed that N1303K and the polymorphisms associated with cis induce weak abnormal splicing and a modification of the quality and the quantity of CFTR protein. These different associations of identified polymorphisms with N1303K in cis could have an impact on the severity of the disease.

A homozygous stop gain mutation in BOD1 gene in a Lebanese patient with syndromic intellectual disability.

Intellectual disability (ID) is a neurodevelopmental disorder characterized by limitations in both intellectual and behavioral functioning. It can occur in non-syndromic and syndromic forms involving multiple organs. While the majority of genetic variants linked to ID are de novo, inherited variants are also detected in some forms. Here, we report a consanguineous Lebanese family presenting with an autosomal recessive syndromic ID characterized by neurodevelopmental delay, mild dysmorphic features, hearing impairment and endocrine dysfunction. Whole exome sequencing enabled the detection of the homozygous nonsense mutation in BOD1, p.R151X, in the proband. BOD1 is required for chromosomes biorientation during cell division. It also contributes to the regulation of cell survival and to the modulation of fatty acid metabolism. Another nonsense mutation in BOD1 was linked to ID in a consanguineous Iranian family. This is the second report of BOD1 mutations in humans and the first in a syndromic ID including gonadal dysfunction and high-frequency hearing impairment. Our findings confirm the involvement of BOD1 in cognitive functioning and expand the clinical spectrum of BOD1 deficiency.

First molecular study in Lebanese patients with Cockayne syndrome and report of a novel mutation in ERCC8 gene.

BACKGROUND: Cockayne Syndrome (CS) is a rare autosomal recessive disorder characterized by neurological and sensorial impairment, dwarfism, microcephaly and photosensitivity. CS is caused by mutations in ERCC6 (CSB) or ERCC8 (CSA) genes. METHODS: Three patients with CS were referred to the Medical Genetics Unit of Saint Joseph University. Sanger sequencing of both ERCC8 and ERCC6 genes was performed: ERCC8 was tested in all patients while ERCC6 in one of them. RESULTS: Sequencing led to the identification of three homozygous mutations, two in ERCC8 (p.Y322* and c.843 + 1G > C) and one in ERCC6 (p.R670W). All mutations were previously reported as pathogenic except for the c.843 + 1G > C splice site mutation in ERCC8 which is novel. CONCLUSIONS: Molecular diagnosis was established in all patients included in our study. A genotype-phenotype correlation is discussed and a link, between mutations and some specific religious communities in Lebanon, is suggested.

BBS10 encodes a vertebrate-specific chaperonin-like protein and is a major BBS locus.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous ciliopathy. Although nine BBS genes have been cloned, they explain only 40-50% of the total mutational load. Here we report a major new BBS locus, BBS10, that encodes a previously unknown, rapidly evolving vertebrate-specific chaperonin-like protein. We found BBS10 to be mutated in about 20% of an unselected cohort of families of various ethnic origins, including some families with mutations in other BBS genes, consistent with oligogenic inheritance. In zebrafish, mild suppression of bbs10 exacerbated the phenotypes of other bbs morphants.

POLD3 haploinsufficiency is linked to non-syndromic sensorineural adult-onset progressive hearing and balance impairments.

Hearing impairment (HI) is a significant health concern globally, influenced by genetic and environmental factors. We had identified a homozygous pathogenic variant in POLD3 in a Lebanese patient with an autosomal congenital recessive syndromic hearing loss (MIM#620869). This variant was found at heterozygous state in the parents, who developed progressive hearing impairment around age 40. We conducted a thorough clinical and genetic assessment of sixteen family members, including physical exams, audiometry and vestibular function evaluations. Additionally, gene expression analysis of the Pold3 gene was performed in mice using RNAscope. Twelve individuals were heterozygous for the variant in POLD3, of whom eight showed bilateral adult-onset HI, typically starting around ages 40-50, and two older patients displaying unilateral vestibular weakness. Additionally, two carriers of the variant developed cancer at an early age. RNAscope confirmed Pold3 expression in auditory and vestibular neurons. Exome sequencing analysis excluded the presence of pathogenic variants in any known hearing impairment or cancer predisposition genes. We present herein, for the first time, evidence of a heterozygous pathogenic POLD3 variant associated with a novel form of autosomal dominant progressive adult-onset hearing and vestibular impairments. We also highlight the necessity for further exploration of the role of POLD3 in cancer predisposition.

Clinical and molecular findings in three Lebanese families with Bietti crystalline dystrophy: report on a novel mutation.

PURPOSE: Bietti crystalline dystrophy (BCD) is a rare autosomal recessive disorder caused by mutation of the cytochrome P450, family 4, subfamily V, polypeptide 2 (CYP4V2) gene and characterized by retinal pigmentary abnormalities and scattered deposits of crystals in the retina and the marginal cornea. The aim of this study was to investigate the spectrum of mutations in CYP4V2 in Lebanese families, and to characterize the phenotype of patients affected with BCD. METHODS: Nine patients from three unrelated Lebanese families were clinically and molecularly investigated. Detailed characterization of the patients' phenotype was performed with comprehensive ophthalmic examination, color vision study, fundus photography, visual field testing, retinal fluorescein angiography, electroretinography, and electrooculography. One family was followed for 12 years. The 11 exons of the CYP4V2 gene were sequenced. RESULTS: Symptoms consisting of night blindness, loss of paracentral visual field, and disturbed color vision were apparent during the third decade of life. Ophthalmoscopy revealed posterior pole crystalline deposits and areas of retinal pigment epithelium atrophy. Fluorescein angiography disclosed geographic areas of the pigment epithelium layer and choriocapillaris atrophy in the posterior pole and fundus periphery. The most striking findings were those of normal electroretinographic responses in some patients and clinical heterogeneity. Two mutations in CYP4V2 were found: p.I111T (c.332T>C) in exon 3 in two families and the novel p.V458M (c.1372G>A) mutation in exon 9 in one family. CONCLUSIONS: These patients are affected with Bietti crystalline dystrophy without corneal involvement. Variation in disease severity and electroretinographic responses suggests that environmental or additional genetic factors influence the course of the retinal disease. The CYP4V2 p.I111T (c.332T>C) mutant allele may be especially prevalent among patients with BCD in Lebanon, resulting from a single founder.

Ambiguous genitalia, microcephaly, seizures, bone malformations, and early death: a distinct MCA/MR syndrome.

We report on two siblings with hypotonia, ambiguous genitalia, microcephaly, ptosis, microretrognathia, thin lips, seizures, absent ossification of pubic rami, and brain abnormalities at the MRI. The two siblings died at 5 and 8 months, respectively. Molecular analysis indicated that SOX9, ARX, and DHCR7 genes were normal. Comparative genomic hybridization (CGH)-array analysis performed on the younger boy indicated two notable deletions, one on paternally inherited chromosome 4, and one on maternally inherited chromosome 5. The same deletions were found in a normal sister. Differential diagnoses and the possibility of a hitherto unreported syndrome are discussed.

The molecular basis of familial hypercholesterolemia in Lebanon: spectrum of LDLR mutations and role of PCSK9 as a modifier gene.

Autosomal dominant hypercholesterolemia (ADH), a major risk for coronary heart disease, is associated with mutations in the genes encoding the low-density lipoproteins receptor (LDLR), its ligand apolipoprotein B (APOB) or PCSK9 (Proprotein Convertase Subtilin Kexin 9). Familial hypercholesterolemia (FH) caused by mutation in the LDLR gene is the most frequent form of ADH. The incidence of FH is particularly high in the Lebanese population presumably as a result of a founder effect. In this study we characterize the spectrum of the mutations causing FH in Lebanon: we confirm the very high frequency of the LDLR p.Cys681X mutation that accounts for 81.5 % of the FH Lebanese probands recruited and identify other less frequent mutations in the LDLR. Finally, we show that the p.Leu21dup, an in frame insertion of one leucine to the stretch of 9 leucines in exon 1 of PCSK9, known to be associated with lower LDL-cholesterol levels in general populations, is also associated with a reduction of LDL-cholesterol levels in FH patients sharing the p.C681X mutation in the LDLR. Thus, by studying for the first time the impact of PCSK9 polymorphism on LDL-cholesterol levels of FH patients carrying a same LDLR mutation, we show that PCSK9 might constitute a modifier gene in familial hypercholesterolemia.

The added value of WES reanalysis in the field of genetic diagnosis: lessons learned from 200 exomes in the Lebanese population.

BACKGROUND: The past few decades have witnessed a tremendous development in the field of genetics. The implementation of next generation sequencing (NGS) technologies revolutionized the field of molecular biology and made the genetic information accessible at a large scale. However, connecting a rare genetic variation to a complex phenotype remains challenging. Indeed, identifying the cause of a genetic disease requires a multidisciplinary approach, starting with the establishment of a clear phenotype with a detailed family history and ending, in some cases, with functional assays that are crucial for the validation of the pathogenicity of a mutation. METHODS: Two hundred Lebanese patients, presenting a wide spectrum of genetic disorders (neurodevelopmental, neuromuscular or metabolic disorders, etc.), sporadic or inherited, dominant or recessive, were referred, over the last three and a half years, to the Medical Genetics Unit (UGM) of Saint Joseph University (USJ). In order to identify the genetic basis of these diseases, Whole Exome Sequencing (WES), followed by a targeted analysis, was performed for each case. In order to improve the genetic diagnostic yield, WES data, generated during the first 2 years of this study, were reanalyzed for all patients who were left undiagnosed at the genetic level. Reanalysis was based on updated bioinformatics tools and novel gene discoveries. RESULTS: Our initial analysis allowed us to identify the specific genetic mutation causing the disease in 49.5% of the cases, in line with other international studies. Repeated WES analysis enabled us to increase the diagnostics yield to 56%. CONCLUSION: The present article reports the detailed results of both analysis and pinpoints the contribution of WES data reanalysis to an efficient genetic diagnosis. Lessons learned from WES reanalysis and interpretation are also shared.

Atypical pyridoxine dependent epilepsy resulting from a new homozygous missense mutation, in ALDH7A1.

Pyridoxine dependent epilepsy (PDE) is a rare autosomal recessive neurometabolic disorder. In the classical form, seizures are observed within the first month of life, while in the atypical form seizures appear later in life, sometimes as late as at the age of 3 years of life. Both types are unresponsive to conventional anticonvulsant therapy, but can be controlled with pyridoxine monotherapy. Mutations in the ALDH7A1 gene, encoding α-aminoadipic semialdehyde dehydrogenase have been reported to cause this disease in most patients. Here, we report on a boy with developmental delay, dysmorphic facial features, and uncontrolled episodes of seizures that appeared at the age of 18 months. By whole exome sequencing (WES) a homozygous missense mutation in ALDH7A1 (NM_001182: c.239T > G, p.V80G) was found. We discuss the importance of WES in such atypical cases.

Pitfalls of homozygosity mapping: an extended consanguineous Bardet-Biedl syndrome family with two mutant genes (BBS2, BBS10), three mutations, but no triallelism.

The extensive genetic heterogeneity of Bardet-Biedl syndrome (BBS) is documented by the identification, by classical linkage analysis complemented recently by comparative genomic approaches, of nine genes (BBS1-9) that account cumulatively for about 50% of patients. The BBS genes appear implicated in cilia and basal body assembly or function. In order to find new BBS genes, we performed SNP homozygosity mapping analysis in an extended consanguineous family living in a small Lebanese village. This uncovered an unexpectedly complex pattern of mutations, and led us to identify a novel BBS gene (BBS10). In one sibship of the pedigree, a BBS2 homozygous mutation was identified, while in three other sibships, a homozygous missense mutation was identified in a gene encoding a vertebrate-specific chaperonine-like protein (BBS10). The single patient in the last sibship was a compound heterozygote for the above BBS10 mutation and another one in the same gene. Although triallelism (three deleterious alleles in the same patient) has been described in some BBS families, we have to date no evidence that this is the case in the present family. The analysis of this family challenged linkage analysis based on the expectation of a single locus and mutation. The very high informativeness of SNP arrays was instrumental in elucidating this case, which illustrates possible pitfalls of homozygosity mapping in extended families, and that can be explained by the rather high prevalence of heterozygous carriers of BBS mutations (estimated at one in 50 in Europeans).

Familial Mediterranean fever (FMF) in Lebanon and Jordan: a population genetics study and report of three novel mutations.

Familial Mediterranean fever (FMF) is an autosomal recessive disease mostly frequent in Mediterranean populations. Over 50 mutations have been identified in the gene responsible for the disease, MEFV. The present study reports the frequencies of MEFV mutations in 558 Lebanese and 55 Jordanian FMF patients and points out the severity of the M694V frequently observed mutation among these patients. Three novel mutations, T177I, S108R and E474K were also identified in the Lebanese group. An excess of homozygotes and a deficit of heterozygotes were observed in both samples when compared to the expected number of observed genotypes under the Hardy-Weinberg hypothesis. Homozygotes for M694V and M694I were still in excess in the Lebanese group of patients, even after consanguinous homozygotes were removed, or population structure was considered. This excess is therefore neither due to consanguinity nor to subgroups in the Lebanese population, but rather to more remote consanguinity or to a selection bias favoring the census of these genotypes. The fact that FMF female patients were less censed than male patients may be due to the greater resistance of females to pain and to the possibility of confusing abdominal and gynecological pain. The phenotypic heterogeneity of the FMF could then originate both from genetic causes like allelic heterogeneity or modulating genes, and cultural background facing the physiological consequences of genotypes at risk.

N1303K (c.3909C>G) mutation and splicing: implication of its c.[744-33GATT(6); 869+11C>T] complex allele in CFTR exon 7 aberrant splicing.

Cystic Fibrosis is the most common recessive autosomal rare disease found in Caucasians. It is caused by mutations on the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that encodes a protein located on the apical membrane of epithelial cells. c.3909C>G (p.Asn1303Lys, old nomenclature: N1303K) is one of the most common worldwide mutations. This mutation has been found at high frequencies in the Mediterranean countries with the highest frequency in the Lebanese population. Therefore, on the genetic level, we conducted a complete CFTR gene screening on c.3909C>G Lebanese patients. The complex allele c.[744-33GATT(6); 869+11C>T] was always associated with the c.3909C>G mutation in cis in the Lebanese population. In cellulo splicing studies, realized by hybrid minigene constructs, revealed no impact of the c.3909C>G mutation on the splicing process, whereas the associated complex allele induces minor exon skipping.

Contribution of copy number variants (CNVs) to congenital, unexplained intellectual and developmental disabilities in Lebanese patients.

BACKGROUND: Chromosomal microarray analysis (CMA) is currently the most widely adopted clinical test for patients with unexplained intellectual disability (ID), developmental delay (DD), and congenital anomalies. Its use has revealed the capacity to detect copy number variants (CNVs), as well as regions of homozygosity, that, based on their distribution on chromosomes, indicate uniparental disomy or parental consanguinity that is suggestive of an increased probability of recessive disease. RESULTS: We screened 149 Lebanese probands with ID/DD and 99 healthy controls using the Affymetrix Cyto 2.7 M and SNP6.0 arrays. We report all identified CNVs, which we divided into groups. Pathogenic CNVs were identified in 12.1% of the patients. We review the genotype/phenotype correlation in a patient with a 1q44 microdeletion and refine the minimal critical regions responsible for the 10q26 and 16q monosomy syndromes. Several likely causative CNVs were also detected, including new homozygous microdeletions (9p23p24.1, 10q25.2, and 8p23.1) in 3 patients born to consanguineous parents, involving potential candidate genes. However, the clinical interpretation of several other CNVs remains uncertain, including a microdeletion affecting ATRNL1. This CNV of unknown significance was inherited from the patient's unaffected-mother; therefore, additional ethnically matched controls must be screened to obtain enough evidence for classification of this CNV. CONCLUSION: This study has provided supporting evidence that whole-genome analysis is a powerful method for uncovering chromosomal imbalances, regardless of consanguinity in the parents of patients and despite the challenge presented by analyzing some CNVs.

Homozygous mutation of the IGF1 receptor gene in a patient with severe pre- and postnatal growth failure and congenital malformations.

BACKGROUND: Heterozygous mutations in the IGF1 receptor (IGF1R) gene lead to partial resistance to IGF1 and contribute to intrauterine growth retardation (IUGR) with postnatal growth failure. To date, homozygous mutations of this receptor have not been described. SUBJECT: A 13.5-year-old girl born from healthy first-cousin parents presented with severe IUGR and persistent short stature. Mild intellectual impairment, dysmorphic features, acanthosis nigricans, and cardiac malformations were also present. METHODS: Auxological and endocrinological profiles were measured. All coding regions of the IGF1R gene including intron boundaries were amplified and directly sequenced. Functional characterization was performed by immunoblotting using patient's fibroblasts. RESULTS: IGF1 level was elevated at 950NG/ML (+7 S.D.). Fasting glucose level was normal associated with high insulin levels at baseline and during an oral glucose tolerance test. Fasting triglyceride levels were elevated. sequencing of the IGF1R gene led to the identification of a homozygous variation in exon 2: c.119G>T (p.Arg10Leu). As a consequence, IGF1-dependent receptor autophosphorylation and downstream signaling were reduced in patient's fibroblasts. Both parents were heterozygous for the mutation. CONCLUSION: The homozygous mutation of the IGF1R is associated with severe IUGR, dysmorphic features, and insulin resistance, while both parents were asymptomatic heterozygous carriers of the same mutation.

Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: identification of two novel mutations, and description of a founder effect.

Progressive pseudorheumatoid dysplasia (PPD) is a rare autosomal recessive syndrome characterized by the presence of spondyloepiphyseal dysplasia associated with pain, stiffness, and swelling of multiple joints, osteoporosis, and the absence of destructive bone changes. The disorder is caused by mutations of the WISP3 gene located on chromosome 6q22. We hereby report the molecular study of the WISP3 gene in nine unrelated consanguineous families originating from the Middle-East: three from Lebanon, five from Syria, and one from Palestinian Bedouin descent, all affected with PPD. Five different sequence variations were identified in the WISP3 gene, two of them being new mutations: the c.589G --> C transversion at codon 197, responsible for a splicing defect (A197fsX201); and the c.536_537delGT deletion (C179fsX), both in exon 3. In all other families, the affected patients were homozygous for a previously described nonsense mutation, namely c.156C --> A (C52X). Interestingly, in the latter families, the C52X mutation was always found associated with a novel c.248G --> A (G83E) variation, suggesting the existence of a founder effect.