Fetal akinesia deformation sequence syndrome associated with recessive TTN variants

Arthrogryposis multiplex congenita (AMC) [also known as multiple joints contracture or Fetal Akinesia Deformation Sequence (FADS)] is etiologically a heterogeneous condition with an estimated incidence of approximately 1 in 3000 live births and much higher incidence when prenatally diagnosed cases are included. The condition can be acquired or secondary to fetal exposures and can also be caused by a variety of single-gene disorders affecting the brain, spinal cord, peripheral nerves, neuromuscular junction, muscle, and a variety of disorders affecting the connective tissues (Niles et al., Prenatal Diagnosis, 2019; 39:720–731). The introduction of next-generation gene sequencing uncovered many genes and causative variants of AMC but also identified genes that cause both dominant and recessive inherited conditions with the variability of clinical manifestations depending on the genes and variants. Molecular diagnosis in these cases is not only important for prognostication but also for the determination of recurrence risk and for providing reproductive options including preimplantation and prenatal diagnosis. TTN, the largest known gene in the human genome, has been known to be associated with autosomal dominant dilated cardiomyopathy. However, homozygote and compound heterozygote pathogenic variants with recessive inheritance have rarely been reported. We report the effect of recessive variants located within the fetal IC and/or N2BA isoforms in association with severe FADS in three families. All parents were healthy obligate carriers and none of them had cardiac or skeletal muscle abnormalities. This report solidifies FADS as an alternative phenotypic presentation associated with homozygote/compound heterozygous pathogenic variants in the TTN.     

A novel PAX1 null homozygous mutation in autosomal recessive otofaciocervical syndrome associated with severe combined immunodeficiency

Otofaciocervical syndrome (OFCS) is a rare disorder characterized by facial anomalies, cup‐shaped low‐set ears, preauricular fistulas, hearing loss, branchial defects, skeletal anomalies, and mild intellectual disability. Autosomal dominant cases are caused by deletions or point mutations of EYA1. A single family with an autosomal recessive form of OFCS and a homozygous missense mutation in PAX1 gene has been described. We report whole exome sequencing of 4 members of a consanguineous family in which 2 children, showing features of OFCS, expired from severe combined immunodeficiency (SCID). To date, the co‐occurrence of OFCS and SCID has never been reported. We found a nonsense homozygous mutation in PAX1 gene in the 2 affected children. In mice, Pax1 is required for the formation of specific skeletal structures as well as for the development of a fully functional thymus. The mouse model strongly supports the hypothesis that PAX1 depletion in our patients caused thymus aplasia responsible for SCID. This report provides evidence that bi‐allelic null PAX1 mutations may lead to a multi‐system autosomal recessive disorders, where SCID might represent the main feature.     

Identification of 45 novel mutations in the nebulin gene associated with autosomal recessive nemaline myopathy

Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder of skeletal muscle caused by mutations in at least five different genes encoding thin filament proteins of the striated muscle sarcomere. We have previously described 18 different mutations in the last 42 exons of the nebulin gene (NEB) in 18 families with NM. Here we report 45 novel NEB mutations detected by denaturing high-performance liquid chromatography (dHPLC) and sequence analysis of all 183 NEB exons in NM patients from 44 families. Altogether we have identified, including the deletion of exon 55 identified in the Ashkenazi Jewish population, 64 different mutations in NEB segregating with autosomal recessive NM in 55 families. The majority (55%) of the mutations in NEB are frameshift or nonsense mutations predicted to cause premature truncation of nebulin. Point mutations (25%) or deletions (3%) affecting conserved splice signals are predicted in the majority of cases to cause in-frame exon skipping, possibly leading to impaired nebulin-tropomyosin interaction along the thin filament. Patients in 18 families had one of nine missense mutations (14%) affecting conserved amino acids at or in the vicinity of actin or tropomyosin binding sites. In addition, we found the exon 55 deletion in four families. The majority of the patients (in 49/55 families) were shown to be compound heterozygous for two different mutations. The mutations were found in both constitutively and alternatively expressed exons throughout the NEB gene, and there were no obvious mutational hotspots. Patients with more severe clinical pictures tended to have mutations predicted to be more disruptive than patients with milder forms.     

Severe combined immunodeficiency syndrome associated with autosomal recessive familial multiple gastrointestinal atresias: study of a family

Hereditary multiple atresias involving the gastrointestinal tract from pylorus to rectum are the most unusual form of intestinal atresia; the type of inheritance was suggested to be autosomal recessive. The inheritance of the severe combined immunodeficiency syndrome can be autosomal recessive or X-linked. We report on 3 sibs with multiple-level intestinal atresias. One sib had severe combined immunodeficiency syndrome and clinical histories of the other 2 sibs strongly suggested a congenital immunodeficiency syndrome. The parents of those children were healthy and nonconsanguineous. To our knowledge, this is the first report of the association of multiple gastrointestinal atresias and immunodeficiency which appears to have an autosomal recessive pattern of transmission. Our family report suggests that, in the presence of multiple gastrointestinal atresias, attention should be given to possible associated immunological disorders.     

Combined immunodeficiency phenotype associated with inappropriate spontaneous and activation-induced apoptosis

Programmed death of T cells has been proposed as one of the mechanisms by which HIV induces a decline in the number and functions of T cells in advanced AIDS. In this study we report on a patient affected by a congenital form of combined immunodeficiency presenting as a profound T cell activation deficiency. Subsequently, a gradual loss of T cells occurred, eventually resulting in a classical form of severe combined immunodeficiency (SCID). In this patient a sizeable fraction of apoptotic cells was documented in the first phase of the disease by either propidium iodide staining or DNA fragmentation analysis. The presence of anergic T cells of maternal origin and engrafted in the child was excluded by analysis of DNA polymorphic regions. At 4 years of age the patient died of disseminated interstitial pneumopathy, while still awaiting an HLA-matched bone marrow transplantation. On the occasion of a new pregnancy in the mother, the prenatal immunological evaluation of the female fetus revealed a T⁻ B⁺ SCID phenotype. This is the first observation of a primary immunodeficiency associated with inappropriate apoptosis.     

Mutations in the mitochondrial gene C12ORF65 lead to syndromic autosomal recessive intellectual disability and show genotype phenotype correlation

Homozygosity mapping and exome sequencing in two affected siblings of a consanguineous family with mild intellectual disability, spastic paraplegia, and strabismus revealed a homozygous premature stop mutation at codon 139 of C12ORF65. Two previous studies reported truncating mutations at positions 84 and 132 of the protein. However, symptoms of the referred patients were only partially overlapping. Considering our findings, we now conclude that truncating mutations in C12ORF65 lead to a variable phenotype with intellectual disability, spastic paraplegia, and ophthalmoplegia as common symptoms. Further, we confirm a genotype–phenotype correlation between increasing length of the truncated protein and decreasing severity of symptoms.     

Complete arrest from pro- to pre-B cells in a case of B cell-negative severe combined immunodeficiency (SCID) without recombinase activating gene (RAG) mutations

The B-cell lineage in a patient with B-cell-negative severe combined immunodeficiency (SCID) was analysed by using antisurrogate light chain (SL) MoAbs. Peripheral CD3(+) T cells and CD19(+) B cells were absent in the patient. The common gamma (gamma c) chain was expressed normally on the patient's peripheral NK cells and his peripheral mononuclear cells did not possess any mutations in recombinase activating gene (RAG)-1, 2. Normal levels of expression of Ku70 and Ku80 protein were found by Western blot analysis. The patient did, however, display an increase in fibroblast sensitivity to irradiation. Furthermore, flow cytometric analyses of bone marrow cells showed that surface IgM and cytoplasmic mu positive cells were absent and that CD19(+) B cells were composed of only CD34(+) terminal deoxynucleotidyl transferase (TdT)(+) SL(+) pro-B cells. The complete arrest of pro- to pre-B cell development in the SCID patient's bone marrow suggests that some genes involved in V(D)J recombination, excepting the RAG gene, may play a causative role in the immunodeficiency.     

Mutations at Ser331 in the HSN type I gene SPTLC1 are associated with a distinct syndromic phenotype

Mutations in the serine palmitoyltransferase subunit 1 (SPTLC1) gene are the most common cause of hereditary sensory neuropathy type 1 (HSN1). Here we report the clinical and molecular consequences of a particular mutation (p.S331Y) in SPTLC1 affecting a patient with severe, diffuse muscle wasting and hypotonia, prominent distal sensory disturbances, joint hypermobility, bilateral cataracts and considerable growth retardation. Normal plasma sphingolipids were unchanged but 1-deoxy-sphingolipids were significantly elevated. In contrast to other HSN patients reported so far, our findings strongly indicate that mutations at amino acid position Ser331 of the SPTLC1 gene lead to a distinct syndrome.     

Connexin26 mutations associated with the most common form of non- syndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans

Non-syndromic neurosensory autosomal recessive deafness (NSRD) is the most common form of genetic hearing loss. Previous studies defined at least 15 human NSRD loci. Recently we demonstrated that DFNB1, located on the long arm of chromosome 13, accounts for approximately 80% of cases in the Mediterranean area. Further analysis with additional markers now identifies several recombinants which narrow the candidate region to approximately 5 cM, encompassed by markers D13S141 and D13S232 and including several ESTs and candidate genes, including the connexin26 (GJB2) gene. Analysis of PCR products from our affected patients' DNA shows two frameshift mutations in the connexin26 gene. Deletion of a G within a stretch of six Gs at position 35 of the GJB2 cDNA (mutation 35delG) leads to premature chain termination and is present in 63% of NSRD chromosomes, demonstrating linkage to chromosome 13. Deletion of a T at position 167 of GJB2 (mutation 167delT), also resulting in premature chain termination, was detected in another patient. Four neutral sequence polymorphisms were also identified. These findings are in agreement with a recent study showing that mutations in the connexin26 gene are associated with genetic forms of deafness in three Pakistani families and that GJB2 is DFNB1. Connexin26 is a member of a large family of proteins involved in formation of gap junctions, which are involved in electrical synapses and the direct transfer of small molecules and ionic currents between neighboring cells. The identification of GJB2 as the DFNB1 gene should provide a better understanding of the biology of normal and abnormal hearing, help form the basis for diagnosis and may facilitate development of strategies for treatment of this common genetic disorder.      

B-cell maturation defects in common variable immunodeficiency and association with clinical features

AIMS: Patients with common variable immunodeficiency (CVID) often have defects in post-antigenic B-cell differentiation with fewer memory B cells and impaired isotype switching. We aimed to classify CVID patients according to these defects and determine whether this predicted clinical manifestations. METHODS: We analysed the memory marker CD27, maturation marker CD21, and IgD on peripheral blood B cells from 31 CVID patients and 23 controls using a whole-blood lysis technique, allocated patients according to two classifications ('Freiburg' and 'Paris') and correlated results with clinical manifestations. RESULTS: CVID patients had fewer memory (CD27(+)) B cells and isotype-switched (IgD(-)) memory B cells in absolute number and proportion. Many CVID patients had increased immature (CD21(-)) B cells. Lymphoproliferation and autoimmune cytopenias were found almost exclusively in these patients, including Freiburg group Ia (decreased switched memory and increased immature B cells), but also those with normal switched memory and increased immature B cells. The Paris classification was less useful in predicting clinical manifestations. CONCLUSIONS: CVID is associated with defects in memory B-cell differentiation. Subclassification helps identify patients with clinical manifestations, particularly lymphoproliferation and autoimmune cytopenias in those with impaired B-cell maturation and isotype switching. Routine B-cell phenotyping may assist clinicians in predicting these clinical features.     

B cells from a distinct subset of patients with common variable immunodeficiency (CVID) have increased CD95 (Apo-1/fas), diminished CD38 expression, and undergo enhanced apoptosis.

We investigated the role of apoptosis in the differentiation failure of B cells from a selected subpopulation of patients with CVID delineated by B cell surface marker analysis, in vitro IgE response, and molecular markers of B cell VH gene repertoire. These patients had altered display of B cell surface molecules that play a role in apoptosis. The patients' B cells had a 4.5-250-fold increase in CD95 (Apo-1, fas) expression and increased CD95 display on their T cells. CD38, a molecule important in preventing germinal centre B cell apoptosis, was reduced on the patients' B cells. The expression of this molecule was inducible on the CVID lymphocytes with retinoic acid. Increased spontaneous apoptosis in vitro was observed with the patients' B (23%) and T cells (10%) compared with normal cells (13% and 3%, respectively). Stimulation in vitro with IL-4 and CD40 rescued the B cells from apoptosis and allowed for their differentiation. However, IL-4 plus alpha CD40-driven immunoglobulin production was not quantitatively or qualitatively normal. Failure to overcome apoptosis, a normal step in germinal centre B cell development, may be involved in the lack of differentiation seen in this subset of CVID patients.     

A unique syndrome of immunodeficiency and autoimmunity associated with absent T cell CD2 expression

CD2 is a glycoprotein expressed on the surface of human T cells that mediates adhesion between T cells and antigen presenting cells. CD2 also functions in concert with the T cell receptor to transduce signals that lead to T cell activation. The CD8 and CD4 molecules are transmembrane glycoproteins that are expressed on mutually exclusive populations of mature T cells and bind to determinants on major histocompatibility complex class I and class II molecules respectively. Like CD2, CD4 and CD8 function to promote adhesion between T cells and antigen presenting cells and potentiate signaling via the T cell receptor. We studied a patient with idiopathic lymphopenia and disseminated infection withMycobacterium avium. The patient also suffered from recurrent deep venous thrombosis in association with anticardiolipin and anti-DNA antibodies. Peripheral blood T cells from this patient were polyclonal and expressed no detectable CD2 RNA or protein as determined by northern blotting, immunofluorescent staining with anti-CD2 antibodies, and failure to form rosettes with sheep red blood cells. In addition, the majority (85%) of this patient's T cells did not express either CD4 or CD8 but did express the / T cell receptor. T cells from this patient failed to respond to stimulation with alloantigen or specific antigen. In contrast, there was a normal response to stimulation with immobilized anti-CD3 antibody. The clinical and immunologic findings in this patient providein vivo evidence that the accessory molecules CD2, CD4, and CD8 play important roles in the regulation of normal human T cell activation.     

Primrose syndrome associated with unclassified immunodeficiency and a novel ZBTB20 mutation

Primrose syndrome is a congenital malformation syndrome characterized by intellectual disability, developmental delay, progressive muscle wasting, and ear lobe calcification. Mutations in the ZBTB20 gene have been established as being accountable for this syndrome. In this study, a novel de novo ZBTB20 mutation, NM_001164342.2:c.1945C>T (p.Leu649Phe), has been identified through whole exome sequencing (WES) in a female patient presenting a typical Primrose phenotype. Because the present patient exhibited recurrent otitis media, detailed immunological examinations were performed in this study and subnormal immunoglobulin levels were firstly identified in a Primrose patient. Anatomical anomaly of the inner ear has never been reported in this patient and WES data did not include any relevant variants causally linked with the immunologic defect. Thus, there is a possibility of a relation between an unclassified immunodeficiency with selective IgG2 deficiency and Primrose syndrome and this may be the reason of recurrent otitis media frequently observed in Primrose patients. Because subnormal levels of IgG2 in this patient might be caused by an unrelated and still uncharacterized genetic cause, further studies are required to prove the causal link between aberrant ZBTB20 function and immunodeficiency.     

One gene,two phenotypes: ROR2 mutations in autosomal recessive Robinow syndrome and autosomal dominant brachydactyly type B

Autosomal recessive Robinow syndrome (RRS) is a severe skeletal dysplasia with short stature, generalized limb shortening, segmental defects of the spine, brachydactyly, and a dysmorphic facial appearance. The gene encoding receptor orphan receptor tyrosine kinase 2 (ROR2) is located on chromosome 9q22 and homozygous loss-of-function mutations in this gene are responsible for RRS. Moreover, knocking out the mouse Ror2 gene causes mesomelic dwarfism in the homozygous state, with almost identical features to recessive Robinow syndrome. The protein product of this gene is a cell membrane receptor, containing distinct motifs including an immunoglobulin-like (Ig) domain, a Frizzled-like cysteine-rich domain (FRZ or CRD), and a kringle domain (KD) in the extracellular region; and an intracellular region with tyrosine kinase (TK), serine/threonine-rich, and proline-rich structures. The extracellular motifs of the ROR2 protein are known to be involved in protein-protein interactions. The tyrosine kinase domain is involved in an as yet uncharacterized signaling pathway. Interestingly, heterozygous mutations in ROR2 have recently been shown to give rise to autosomal dominant brachydactyly type B1 (BDB1). This condition is characterized by terminal deficiency of fingers and toes. A variety of mutations have been reported in ROR2. Here, these genetic defects are compiled and possible genotype-phenotype correlations are discussed.     

Variants of the ectodysplasin A1 receptor gene underlying homozygous cases of autosomal recessive hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasia (HED) is a rare genetic condition resulting from defective development of ectodermal derivatives, such as hair, teeth, and sweat glands. Autosomal recessive (AR) forms of HED may be caused by pathogenic variants of the ectodysplasin A1 receptor (EDAR) gene that encodes a receptor involved in the NF-κB signaling pathway. Here, we describe three cases of AR-HED in families of Turkish, Austrian, and German-American origin (with or without known consanguinity). In these cases, two out-of-frame deletions and a pathogenic missense variant of EDAR were found to be disease-causing due to reduced availability of the respective messenger RNA or impaired interaction of the encoded protein with its binding partner leading to diminished signal transduction. The same missense variant, c.1258C>T (p.Arg420Trp), has actually been reported to be restricted to the Icelandic population and to be associated with non-syndromic tooth agenesis but not HED. As our patient has no known relationship to Icelandic individuals and displays a rather severe HED phenotype, we suggest that EDAR-Arg420Trp is a more widespread variant, possibly with variable clinical expressivity.     

A novel compound heterozygous TACI mutation in an autosomal recessive common variable immunodeficiency (CVID) family

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by low immunoglobulin serum levels and increased susceptibility to infections. Underlying genetic causes are only known in less than 15% of patients and encompass mutations in the genes encoding for ICOS, TACI, BAFF-R, CD19, CD20, CD81 and MSH5. TACI is the most frequently mutated gene among CVID patients. We report on two pediatric Italian male siblings with hypogammaglobulinemia and recurrent respiratory and gastrointestinal infections in association with a novel compound heterozygous TACI mutation. Both patients carry the I87N/C104R mutation that has not been reported yet. This results in aberrant TACI expression and abrogates APRIL binding on EBV B cells. This study identifies a novel combined mutation in TNFRSF13B increasing the spectrum of TACI mutations associated with CVID.