Single Kindred Research Articles

Partial penetrance and phenotypic variability of aplasia of lacrimal and salivary glands caused by a novel FGF10 donor splice-site mutation.

Thirteen affected individuals of six generations of a single kindred presented with epiphora evident from infancy. Physical exam and Schirmer test revealed variable expression of tear deficiency, congenital punctal atresia, and dry mouth with multiple caries, without concomitant abnormalities of the ears or digits, commensurate with a diagnosis of aplasia of the lacrimal and salivary glands (ALSG). Reconstruction of the upper lacrimal drainage system was performed in some of the affected individuals. Genetic analysis, testing six affected individuals and three non-affected family members, identified a single novel heterozygous splice-site variant, c.429 + 1, G > T in fibroblast growth factor 10 (FGF10) (NM_004465.1), segregating throughout the family as expected for dominant heredity. RT-PCR assays of HEK-293 cells transfected with wild type or mutant FGF10 demonstrated that the variant causes skipping of Exon 2. Notably, individuals sharing the same variant exhibited phenotypic variability, with unilateral or bilateral epiphora, as well as variable expression of dry mouth and caries. Moreover, one of the variant carriers had no ALSG-related clinical findings, demonstrating incomplete penetrance. While coding mutations in FGF10 are known to cause malformations in the nasolacrimal system, this is the second FGF10 splice-site variant and the first donor-site variant reported to cause ALSG. Thus, our study of a unique large kindred with multiple affected individuals heterozygous for the same FGF10 variant highlights intronic splice-site mutations and phenotypic variability/partial penetrance in ALSG.

Biallelic deleterious germline SH2B3 variants cause a novel syndrome of myeloproliferation and multi-organ autoimmunity.

SH2B3 is a negative regulator of multiple cytokine receptor signalling pathways in haematopoietic tissue. To date, a single kindred has been described with germline biallelic loss-of-function SH2B3 variants characterized by early onset developmental delay, hepatosplenomegaly and autoimmune thyroiditis/hepatitis. Herein, we described two further unrelated kindreds with germline biallelic loss-of-function SH2B3 variants that show striking phenotypic similarity to each other as well as to the previous kindred of myeloproliferation and multi-organ autoimmunity. One proband also suffered severe thrombotic complications. CRISPR-Cas9 gene editing of zebrafish sh2b3 created assorted deleterious variants in F0 crispants, which manifest significantly increased number of macrophages and thrombocytes, partially replicating the human phenotype. Treatment of the sh2b3 crispant fish with ruxolitinib intercepted this myeloproliferative phenotype. Skin-derived fibroblasts from one patient demonstrated increased phosphorylation of JAK2 and STAT5 after stimulation with IL-3, GH, GM-CSF and EPO compared to healthy controls. In conclusion, these additional probands and functional data in combination with the previous kindred provide sufficient evidence for biallelic homozygous deleterious variants in SH2B3 to be considered a valid gene-disease association for a clinical syndrome of bone marrow myeloproliferation and multi-organ autoimmune manifestations.

Phenotypic characterization of autosomal dominant progressive cone dystrophies associated with a heterozygous variant c.2512C>T of GUCY2D gene in a large kindred.

In this study, we described a large family presenting different manifestations of cone dystrophy at different ages associated with GUCY2D gene mutation. Sixty-three individuals of a single kindred, including 23 affected with cone dystrophies, were recruited and received ocular examinations, including best corrected visual acuity, intraocular pressure, slit-lamp biomicroscopy, color fundus photograph (CFP), fundus autofluorescence, optical coherence tomography, fluorescence fundus angiography, color vision testing, full-field electroretinography, and electro-oculogram. Whole exome sequencing (WES) and Sanger sequencing were performed for underlying mutations associated with cone dystrophy. There were 23 affected family members. Clinical analysis showed that the proband and other patients had impaired visual acuity ranging from 20/800 to 20/50 with impaired color vision. Fundus photograph showed retinal pigment epithelium (RPE) granular abnormalities with depressed macular reflex in young patients and macular or retinochoriodal atrophy in older patients. OCT examination confirmed the reduced outer retinal thickness or inner retinal thickness, absence of the ellipsoid zone (EZ) and retinal atrophy to varying degrees. Electroretinography revealed a reduced cone response combined with a relatively maintained rod response. WES and Sanger sequencing revealed a heterozygous variant c.2512C>T in the GUCY2D gene of the affected family members. We reported cone dystrophy in 23 affected individuals in a five-generation family and demonstrated different macular abnormalities in OCT scans and CFP at different ages. The multimodal ocular records in our study provide physicians and ophthalmologists with a better understanding of cone dystrophy associated with GUCY2D mutation.

Biallelic Deleterious Germline SH2B3 Variants Cause a Novel Clinical Syndrome of Myeloproliferation and Multi-Organ Autoimmunity

SH2B3 is a negative regulator of cytokine receptor signalling pathways in hematopoietic tissue. Acquired deleterious SH2B3 variants resulting in increased JAK/STAT signalling are observed in myeloproliferative neoplasms (MPN) however the clinical implications and phenotype of deleterious germline variants in SH2B3 are less clear. A single consanguineous kindred has been previously described with homozygous deleterious germline SH2B3 variants presenting with hepatosplenomegaly, autoimmune hepatitis, developmental delay, autoimmune thyroiditis and acute lymphoblastic leukemia (Perez-Garcia, A., et al, Blood, 2013), however a clinically valid gene-disease pair according to formal criteria has not been met to date for SH2B3. We identified two probands from unrelated kindred with germline homozygous deleterious SH2B3 variants. The first patient presented with splenomegaly, thrombocytosis, neutrophilia and a leukoerythroblastic blood film at 3 months old. He developed alopecia areata at 2 years and autoimmune hypothyroidism at 6 years. He was otherwise developmentally normal without any detected liver abnormalities or thrombotic episodes. Whilst his splenomegaly and neutrophil count gradually improved throughout life, his platelet count remained markedly elevated (~1000x109/L at 18 years of age). Targeted NGS identified a homozygous frameshift variant in SH2B3 (NM_005475.3:c.441_468del; p.(Arg148Profs*40)). Both parents were heterozygous carriers of this variant. The second patient was a male born to consanguineous parents and noted shortly after birth to have isolated splenomegaly. He was diagnosed with autoimmune hypothyroidism and Raynaud syndrome at 12 years and was noted to have a persistent thrombocytosis ranging from 550 - 780 x109/L. He developed autoimmune hepatitis and insulin dependent diabetes at 17 years and around the same time he also suffered a right-sided middle cerebral artery territory infarct with a right carotid artery thrombus. Whole genome sequencing detected a homozygous missense variant in SH2B3 (NM_005475.3:c.1204G>A; p.(Val402Met)) in the patient with both parents being heterozygous carriers. In order to understand these variants further we modelled the human p.(Arg148Profs*40) variant from patient 1 in zebrafish. CRISPR-Cas9 gene editing was targeted to the analogous region of zebrafish sh2b3 to create assorted carboxyl-truncating mutations in F0 crispants. On-target gene-editing was verified by Sanger sequencing and NGS. F0 crispants had a significantly increased number of macrophages and CD41-expressing cells (HSCs and thrombocytes), replicating the patient phenotype. Treatment of the sh2b3 crispant fish with the JAK inhibitor ruxolitinib intercepted the myeloproliferative phenotype. The myeloproliferative phenotype was not observed in stable deletion mutants, likely reflecting genetic compensation not present in crispants. Skin-derived fibroblasts from patient 2 who carried the Val402Met homozygous variant, showed increased phosphorylation of JAK2, STAT5 and STAT3 by immunoblotting after stimulation with IL3, GH, GM-CSF and EPO compared to healthy controls. In conclusion, these newly described kindreds showing striking phenotypic similarity to each other as well as the previously described kindred and in conjunction with our functional data provide sufficient evidence for biallelic homozygous deleterious variants in SH2B3 to be considered a valid gene-disease association with a clinical syndrome of bone marrow myeloproliferation and multi-organ autoimmune manifestations. Notably, neither of our patients had evidence of acute lymphoblastic leukemia or overt hematological malignancy and further affected individuals will need to be described in order to fully understand the breadth of the phenotype. Moreover, our data supports ruxolitinib as being a potential therapeutic option for patients with this rare novel immune dysregulatory syndrome.

Charcot-Marie-Tooth disease due to MORC2 mutations in Spain.

MORC2 mutations have been described as a rare cause of axonal Charcot-Marie-Tooth disease (CMT2Z). The aim of this work was to determine the frequency and distribution of these mutations throughout Spain, toprovide a comprehensivephenotypical description and, if possible, to establish a genotype-phenotype correlation. Retrospectively, data on patients diagnosed with CMT2Z in Spain werecollected and clinical, electrophysiological and muscle imaging information were analysed. Fifteen patients with CMT2Z were identified throughout Spain, seven of them belonging to a single kindred, whilst the rest were sporadic. The most common mutation was p.R252W, and four new mutations were identified. Eleven patients were categorized as having a scapuloperoneal phenotype, with asymmetric muscle weakness, early proximal upper limb involvement and frequent spontaneous muscular activity with distal sensory impairment and pes cavus, whilst two presented with a more classic length dependent sensory motor phenotype. This distinction was corroborated by the distribution of musclefatty infiltration in muscle imaging. Two other patients were classified as having a neurodevelopmental phenotype consisting in congenital or early onset, delay in motor milestones, and global developmental delay in one of them. Nerve conduction studies revealed an unequivocally axonal neuropathy with frequent spontaneous activity, and serum creatine kinase levels were increased in 50% of the patients. MORC2 mutations are a rare cause of CMT in Spain, but in-depth phenotyping reveals a recognizable phenotypic spectrum that will be clinically relevant for future identification of this disease.

Combined CNV, haplotyping and whole exome sequencing enable identification of two distinct novel EYS mutations causing RP in a single inbred tribe.

Whole exome sequencing (WES) has become routine in clinical practice, especially in studies of recessive hereditary diseases in inbred consanguineous families, where homozygosity of a founder mutation is assumed. Multiple members of two consanguineous families of a single Bedouin tribe were diagnosed with apparently autosomal recessive/pseudo-dominant retinitis pigmentosa (RP). Affected individuals exhibited severe visual impairment with nyctalopia, marked constriction of visual fields, markedly reduced and delayed responses on electro-retinography (ERG) and eventual loss of central vision. Combined copy-number variant (CNV) analysis, haplotype reconstruction and WES of the kindred identified two distinct novel mutations in EYS (RP25): a p.(W1817*) nonsense mutation (identified through WES) and a large deletion encompassing 9 of the 43 exons, that was missed by WES and was identified through microarray CNV analysis. Segregation analysis of both mutations demonstrated that all affected individuals were either homozygous for one of the mutations, or compound heterozygous for both. The two mutations are predicted to cause loss of function of the encoded protein and were not present in screening of 200 ethnically-matched controls. Our findings of two distinct mutations in the same gene in a single inbred kindred, identified only through combined WES and microarray CNV analysis, highlight the limitations of either CNV or WES alone, as the heterozygous deletion had normal WES read-depth values. Moreover, they demonstrate pitfalls in homozygosity mapping for disease-causing variant identification in inbred communities.

Dysregulation of interleukin 5 expression in familial eosinophilia.

Familial eosinophilia (FE) is a rare autosomal dominant inherited disorder characterized by the presence of lifelong peripheral eosinophilia (>1500/μL). Mapped to chromosome 5q31-q33, the genetic cause of FE is unknown, and prior studies have failed to demonstrate a primary abnormality in the eosinophil lineage. The aim of this study was to identify the cells driving the eosinophilia in FE. Microarray analysis and real-time PCR were used to examine transcriptional differences in peripheral blood mononuclear cells (PBMC), and in purified cell subsets from affected and unaffected family members belonging to a single large kindred. Cytokine levels in serum and PBMC culture supernatants were assessed by suspension array multiplexed immunoassays. Whereas IL-5 mRNA expression was significantly increased in freshly isolated PBMC from affected family members, this was not accompanied by increased mRNA expression of other Th2 cytokines (IL-4 or IL-13). Serum levels of IL-5 and IL-5 receptor α, but not IgE, were similarly increased in affected family members. Of note, IL-5 mRNA expression was significantly increased in purified CD3+ CD4+, CD14+, CD19+, and ILC2 cells from affected family members, as were IL-5 protein levels in supernatants from both stimulated PBMC and ILC2 cultures. These data are consistent with the hypothesis that the eosinophilia in FE is secondary to dysregulation of IL-5 production in PBMC (and their component subsets).

Novel GUCY2D mutation causes phenotypic variability of Leber congenital amaurosis ina large kindred.

BackgroundLeber congenital amaurosis (LCA) is a severe retinal degenerative disease that manifests as blindness or poor vision in infancy. The purpose of this study was to clinically characterize and identify the cause of disease in a large inbred Bedouin Israeli tribe with LCA.MethodsThirty individuals of a single kindred, including eight affected with LCA, were recruited for this study. Patients’ clinical data and electroretinography (ERG) findings were collected. Molecular analysis included homozygosity mapping with polymorphic markers and Sanger sequencing of candidate genes.ResultsOf the eight affected individuals of the kindred, nystagmus was documented in five subjects and keratoconus in three. Cataract was found in 5 of 16 eyes. Photopic and scotopic ERG performed in 5 patients were extinguished. All affected subjects were nearly blind, their visual acuity ranged between finger counting and uncertain light perception. Assuming autosomal recessive heredity of a founder mutation, studies using polymorphic markers excluded homozygosity of affected individuals at the genomic loci of all previously known genes associated with LCA, except GUCY2D. Sequencing of GUCY2D identified a novel missense mutation (c.2129C>T; p.Ala710Val) resulting in substitution of alanine by valine at position 710 within the protein kinase domain of the retina-specific enzyme guanylate cyclase 1 (GC1) encoded by GUCY2D. Molecular modeling implied that the mutation changes the conformation of the regulatory segment within the kinase styk-domain of GC1 and causes loss of its helical structure, likely inhibiting phosphorylation of threonine residue within this segment, which is needed to activate the catalytic domain of the protein.ConclusionsThis is the first documentation of the p.Ala710Val mutation in GC1 and the second ever described mutation in its protein kinase domain. Our findings enlarge the scope of genetic variability of LCA, highlight the phenotypic heterogeneity found amongst individuals harboring an identical LCA mutation, and possibly provide hope for gene therapy in patients with this congenital blinding disease. As the Bedouin kindred studied originates from Saudi Arabia, the mutation found might be an ancient founder mutation in that large community.

Chronic mucocutaneous candidiasis: characterization of a family with STAT-1 gain-of-function and development of an ex-vivo assay for Th17 deficiency of diagnostic utility.

SummaryChronic mucocutaneous candidiasis (CMC) is characterized by recurrent and persistent superficial infections, with Candida albicans affecting the mucous membranes, skin and nails. It can be acquired or caused by primary immune deficiencies, particularly those that impair interleukin (IL)−17 and IL‐22 immunity. We describe a single kindred with CMC and the identification of a STAT1 GOF mutation by whole exome sequencing (WES). We show how detailed clinical and immunological phenotyping of this family in the context of WES has enabled revision of disease status and clinical management. Together with analysis of other CMC cases within our cohort of patients, we used knowledge arising from the characterization of this family to develop a rapid ex‐vivo screening assay for the detection of T helper type 17 (Th17) deficiency better suited to the routine diagnostic setting than established in‐vitro techniques, such as intracellular cytokine staining and enzyme‐linked immunosorbent assay (ELISA) using cell culture supernatants. We demonstrate that cell surface staining of unstimulated whole blood for CCR6+CXCR3–CCR4+CD161+ T helper cells generates results that correlate with intracellular cytokine staining for IL‐17A, and is able to discriminate between patients with molecularly defined CMC and healthy controls with 100% sensitivity and specificity within the cohort tested. Furthermore, removal of CCR4 and CD161 from the antibody staining panel did not affect assay performance, suggesting that the enumeration of CCR6+CXCR3–CD4+ T cells is sufficient for screening for Th17 deficiency in patients with CMC and could be used to guide further investigation aimed at identifying the underlying molecular cause.

Commentary: Effect of Agalsidase Beta on Corneal Verticillata in Patients with Fabry Disease

In our previous article entitled, Ocular manifestations of Fabry disease within in single kindred, we describe the ocular and systemic manifestations of Fabry disease. Fabry disease is a lysosomal storage disease inherited in an X-linked fashion which results in deficient enzyme levels of alpha-galactosidase A (AGAL), an enzyme important for the breakdown of various glycoproteins in the body. AGAL deficiency prevents the effective metabolism of the glycoprotein globotriaosylceramide (Gb3). Consequently, Gb3 deposits within lysosomes in virtually every tissue in the body including the eyes, kidneys, brain, and heart. Increasing storage of Gb3 and the resultant intrinsic inflammatory reactions lead to increased disease burden in the patient with disease progression. The initial symptoms can include angiokeratomas (telangiectatic cutaneous lesions), acroparesthesia (severe pain in hands and feet), hypohidrosis (inability to sweat), gastrointestinal dysmotility, and various ocular manifestations [1].

Identification of a Novel SLC40A1 Arg88Ile Mutation in a Patient with Familial Iron Overload Treated By Phlebotomy

INTRODUCTIONThe cellular iron exporter ferroportin, encoded by the SLC40A1 gene, plays a key role in systemic iron regulation by mediating the absorption of dietary iron from duodenal enterocytes and the release of macrophage iron stores into the plasma. SLC40A1 mutations result in a clinically heterogeneous iron overload disorder exhibiting autosomal dominant transmission. Mutations that impair iron export function result in a classical ferroportin disease phenotype characterized by hyperferritinemia, normal transferrin saturation, and macrophage iron loading, while mutations that impair the regulation of ferroportin by hepcidin result in a non-classical form of disease exhibiting high transferrin saturation and hepatocellular iron loading. Here we report the clinical phenotype of a male patient with a personal and family history of iron overload who was found to harbor a novel SLC40A1 mutation. CLINICAL HISTORYA 39-year-old male of Italian descent came to clinical attention after laboratory evidence of iron overload was detected at the time of a routine physical exam. Serum ferritin was markedly elevated at 5018 ng/mL, while transferrin iron saturation was within the normal range at 42%. A complete blood count revealed hemoglobin 16.1 g/dL, hematocrit 48.7%, and MCV 96.7 fL. Liver function tests revealed mild elevation of transaminases (AST 50 U/L, ALT 114 U/L). HBV and HCV serologies, as well as an anti-nuclear antibody screen, were negative. Rheumatoid factor, ceruloplasmin, and alpha-fetoprotein were within the normal range. Genetic testing for the HFE C282Y, H63D, and S65C variants was negative. Abdominal ultrasound revealed a somewhat course and echogenic liver. The patient's past medical and surgical histories were non-contributory. Family history was notable for a 63-year-old father with non-HFE hemochromatosis treated by phlebotomy, as well as a possible history of iron overload in the paternal grandmother. There was no known family history of hepatocellular carcinoma. The patient reported consuming ≤ 5 alcoholic beverages per week. Review of systems was negative, and no organomegaly was detected on physical exam. The patient began undergoing phlebotomy approximately every 2 weeks, which now has been well tolerated for almost two years. His most recent ferritin level was within the normal range (328 ng/mL). METHODSUsing genomic DNA extracted from peripheral blood as template, all coding regions and intron-exon boundaries of SLC40A1 were amplified by polymerase chain reaction and analyzed by bidirectional Sanger sequencing. Sequence chromatograms were analyzed using Sequencher software. This study was approved by the Yale University Human Investigation Committee (protocol #010412377). RESULTSA heterozygous single nucleotide substitution (c.263G>T, encoding p.Arg88Ile) was identified in exon 3 of the SLC40A1 gene (nomenclature per Ensembl reference transcript ENST00000261024). Ferroportin is a predicted multipass transmembrane protein, and Arg88 resides between two predicted transmembrane domains. Protein sequence alignments reveal that amino acid 88 is conserved as an arginine in ferroportin homologs in species as evolutionarily distant as Xenopus laevis and Danio rerio, suggesting that this residue is required for normal ferroportin function. The p.Arg88Ile variant has not been reported in the 1000 Genomes Project or the Exome Aggregation Consortium, demonstrating that it is not a polymorphism in the general population. The mutation predictor algorithms PolyPhen2, SIFT, and MutationTaster all strongly predicted this mutation to be damaging. DISCUSSIONThe ferroportin variant detected in this case (p.Arg88Ile) represents the third non-synonymous substitution at ferroportin residue 88 detected in patients with iron overload phenotypes. p.Arg88Gly was identified in a 38-year-old male who became anemic under a phlebotomy program (Cunat S., et al., Clin Chem 2007), while p.Arg88Thr was detected in multiple affected members of a single kindred in whom serial phlebotomy was well tolerated (Bach V, et al. Blood Cells Mol Dis 2006). Collectively, these findings suggest that the particular amino acid substitution at residue 88 may influence the degree of cellular iron sequestration. Future work will assess the effect of the Arg88Ile substitution on ferroportin function. DisclosuresNo relevant conflicts of interest to declare.

Loss of function mutation in a mitochondrial chaperone protein leading to dyregulated ROS production, and autoinflammatory disease in a single kindred

Reactive oxygen species (ROS) are known to have many roles in the propagation of the inflammatory process. Indeed increased leucocyte ROS have been implicated in a number of autoinflammatory diseases (TRAPS, FMF, CAPS and FCAS2).

Inherited IL-17RC deficiency in patients with chronic mucocutaneous candidiasis.

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections of the skin, nail, oral, and genital mucosae with Candida species, mainly C. albicans. Autosomal-recessive (AR) IL-17RA and ACT1 deficiencies and autosomal-dominant IL-17F deficiency, each reported in a single kindred, underlie CMC in otherwise healthy patients. We report three patients from unrelated kindreds, aged 8, 12, and 37 yr with isolated CMC, who display AR IL-17RC deficiency. The patients are homozygous for different nonsense alleles that prevent the expression of IL-17RC on the cell surface. The defect is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. However, in contrast to what is observed for the IL-17RA- and ACT1-deficient patients tested, the response to IL-17E (IL-25) is maintained in these IL-17RC-deficient patients. These experiments of nature indicate that human IL-17RC is essential for mucocutaneous immunity to C. albicans but is otherwise largely redundant.

Genomic structural variants are linked with intellectual disability.

Mutations in more than 500 genes have been associated with intellectual disability (ID) and related disorders of cognitive function, such as autism and schizophrenia. Here we aimed to unravel the molecular epidemiology of non-specific ID in a genetic isolate using a combination of population and molecular genetic approaches. A large multigenerational pedigree was ascertained within a Dagestan Genetic Heritage research program in a genetic isolate of indigenous ethnics. Clinical characteristics of the affected members were based on combining diagnoses from regional psychiatric hospitals with our own clinical assessment, using a Russian translation of the structured psychiatric interviews, the Diagnostic Interview for Genetic Studies and the Family Interview for Genetic Studies, based on DSM-IV criteria. Weber/CHLC 9.0 STRs set was used for multipoint parametric linkage analyses (Simwalk2.91). Next, we checked CNVs and LOH (based on Affymetrix SNP 5.0 data) in the linked with ID genomic regions with the aim to identify candidate genes associated with mutations in linked regions. The number of statistically significant (p ≤ 0.05) suggestive linkage peaks with 1.3 < LOD < 3.0 we detected in a total of 10 genomic regions: 1q41, 2p25.3-p24.2, 3p13-p12.1, 4q13.3, 10p11, 11q23, 12q24.22-q24.31, 17q24.2-q25.1, 21q22.13 and 22q12.3-q13.1. Three significant linkage signals with LOD >3 were obtained at 2p25.3-p24.2 under the dominant model, with a peak at 21 cM flanked by loci D2S2976 and D2S2952; at 12q24.22-q24.31 under the recessive model, with a peak at -120 cM flanked by marker D12S2070 and D12S395 and at 22q12.3 under the dominant model, with a peak at 32 cM flanked by marker D22S683 and D22S445. After a set of genes had been designated as possible candidates in these specific chromosomal regions,we conducted an exploratory search for LOH and CNV based on microarray data to detect structural genomic variants within five ID-linked regions with LOD scores between 2.0 and 3.9. In these selected regions we obtained 173 ROH segments and 98 CN segments. Further analysis of region 2p25.3-p24.2 revealed deletions within genes encoding MYTL, SNTG2 and TPO among five of 21 affected cases at 2p25.3-p24.2. In the ID-linked region at 12q24.22-12q24.31 19 out of 21 ID cases carried segmental CNV and 20 of 21 them displayed ROH segments with mean size lengths for ID cases 2512 kb (500-6,472 kb) and for healthy control 682 kb (531-986 kb), including the genes MED13L, HRK, FBXW8, TESC, CDK2AP1 and SBNO1. Seven of 21 affected pedigree members displayed segmental deletions at 22q12.3 that includes the gene LARGE. Eight affected pedigree members carried ROH segments and 6 CN segments at 10p11.23-p11.21 containing the genes ZEB1, c10orf68 and EPC1. Our linkage and structural genomic variation analyses in a remote highland genetic isolate with aggregation of ID demonstrated that even highly isolated single kindred ID has oligo/polygenic pathogenesis. The results obtained implicate 10 genomic regions linked with ID that contain some of previously reported candidate genes, including HRK, FBXW8, TESC, CDK2AP1 and SBNO1 at 12q24 that were shown in recent studies as associated with brain measures derived from MRI scans.

Identification of a novel monogenic autoinflammatory disease due to mutation in a mitochondrial chaperone protein in a single kindred, and cure with allogeneic haematopoietic stem cell transplantation

The monogenic autoinflammatory syndromes are characterised by seemingly unprovoked inflammation which derives from a disruption of innate immunity. Novel as yet undefined autosomal recessive syndromes are increasingly recognised in consanguineous families. This type of family is ideal for genetic mapping.

Chronic Granulomatous Disease in Morocco: Genetic, Immunological, and Clinical Features of 12 Patients from 10 Kindreds

Chronic granulomatous disease (CGD) is characterized by an inability of phagocytes to produce reactive oxygen species (ROS), which are required to kill some microorganisms. CGD patients are known to suffer from recurrent bacterial and/or fungal infections from the first year of life onwards. From 2009 to 2013, 12 cases of CGD were diagnosed in Morocco. We describe here these Moroccan cases of CGD. We investigated the genetic, immunological and clinical features of 12 Moroccan patients with CGD from 10 unrelated kindreds. All patients were children suffering from recurrent bacterial and/or fungal infections. All cases displayed impaired NADPH oxidase activity in nitroblue tetrazolium (NBT), dihydrorhodamine (DHR) or 2',7' dichlorofluorescein diacetate (DCFH-DA) assays. Mutation analysis revealed the presence of four different mutations of CYBB in four kindreds, a recurrent mutation of NCF1 in three kindreds, and a new mutation of NCF2 in three patients from a single kindred. A large deletion of CYBB gene has detected in a patient. The causal mutation in the remaining one kindred was not identified. The clinical features and infectious agents found in these patients were similar to those in CGD patients from elsewhere. The results of mutation analysis differed between kindreds, revealing a high level of genetic and allelic heterogeneity among Moroccan CGD patients. The small number of patients in our cohort probably reflects a lack of awareness of physicians. Further studies on a large cohort are required to determine the incidence and prevalence of the disease, and to improve the description of the genetic and clinical features of CGD patients in Morocco.

GDF15 Is Not The Sole Negative Regulator Of Hepcidin In Hereditary Xerocytosis

Hereditary xerocytosis (HX) is a rare form of hemolytic anemia with autosomal dominant inheritance in which iron loading is a prominent feature. The mutated gene causing HX has been identified as FAM38A, which codes for the PIEZO1 protein, a mechanosensitive ion channel [1].The phenotype and genotype of HX has been characterized in a large Canadian family with members spanning three generations and seven decades[2]. Affected family members demonstrate fully-compensated hemolytic anemia (average reticulocyte count 9.9%, hemoglobin 135g/L) and their red cells exhibit decreased levels of osmotic fragility. Despite elevated reticulocyte counts and elevated unconjugated bilirubin levels, serum lactate dehydrogenase levels are normal, suggesting that little if any of the erythropoiesis is ‘ineffective'. Affected family members accumulate iron with age, with average ferritin levels for adults of 478 μg/L. The mechanism behind the iron loading in HX is not known. It is now recognized that in these forms of anemia, hepcidin levels are inappropriately low for the degree of iron store, implying the presence of a mediator produced by the hematopoietic progenitors that acts on the liver to suppress hepcidin production. One pathway that appears important in regulating hepcidin synthesis is the bone morphogenetic protein (BMP)-SMAD signaling cascade. The importance of BMP6 is evident from studies using gene knockout mice. Likewise, liver-targeted knockdown of SMAD4 impairs production of hepcidin and resulted in iron overload in the mice [3]. BMP6 may act in an autocrine fashion, and its secretion by hepatocytes is upregulated in the presence of elevated iron levels.Another proposed pathway for hepcidin regulation – speculated to be involved in the erythropoietic regulation of iron – involves another cytokine also from the TGF-β superfamily. Growth differentiation factor 15 (GDF15) is expressed in high levels in placenta tissue and in smaller quantities in the liver, lungs and kidneys. It is also secreted by erythroblasts, at least in culture. Plasma levels of GDF15 are greatly increased in thalassemia and correlate with markers of erythroid mass such as the soluble transferrin receptor. Serum from thalassemic patients suppresses hepcidin mRNA expression by cultured hepatocytes, an effect partially recapitulated by recombinant GDF15, suggesting that the cytokine requires a co-factor for full hepcidin inhibition [4].We evaluated the level of hepcidin, EPOand ferritin along with GDF15 in 29 affected individuals from a single kindred with HX, and a similar number of age matched unaffected family members to explore the putative erythropoietic regulator of iron absorption in a homogeneous genetic context. We find that ferritin level positively predicts hepcidin level (p<.001) and age negatively predicts hepcidin level. After adjustment for age and ferritin, GDF15 does negatively predict hepcidin level(p=.046 in the final fully-adjusted model). However, in a regression model adjusting for ferritin, age and GDF15, xerocytosis still predicts hepcidin level, with lower hepcidin among the affected family members (p<.001).These results suggest that GDF15 may be one mediator of hepcidin suppression and iron loading in hereditary xerocytosis. However, its effect is insufficient to explain the full iron-loading propensity.'1. Zarychanski, R., et al., Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary xerocytosis. Blood, 2012. 120(9): p. 1908-15.2. Houston, B.L., et al., Refinement of the hereditary xerocytosis locus on chromosome 16q in a large Canadian kindred. Blood Cells Mol Dis, 2011. 47(4): p. 226-31.3. Corradini, E., et al., Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice. Hepatology, 2011. 54(1): p. 273-84.4. Tanno, T., et al., High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med, 2007. 13(9): p. 1096-101. Disclosures:No relevant conflicts of interest to declare.

Creatine Kinase Adenosine Triphosphate and Phosphocreatine Energy Supply in a Single Kindred of Patients With Hypertrophic Cardiomyopathy

A lethal and extensively characterized familial form of hypertrophic cardiomyopathy (HC) is due to a point mutation (Arg403Gln) in the cardiac β-myosin heavy chain gene. Although this is associated with abnormal energy metabolism and progression to heart failure in an animal model, in vivo cardiac energetics have not been characterized in patients with this mutation. Noninvasive phosphorus saturation transfer magnetic resonance spectroscopy was used to measure the adenosine triphosphate supplied by the creatine kinase (CK) reaction and phosphocreatine, the heart's primary energy reserve, in 9 of 10 patients from a single kindred with HC caused by the Arg403GIn mutation and 17 age-matched healthy controls. Systolic and diastolic function was assessed by echocardiography in all 10 patients with HC. The patients with HC had impairment of diastolic function and mild systolic dysfunction, when assessed using global systolic longitudinal strain. Myocardial phosphocreatine was significantly decreased by 24% in patients (7.1 ± 2.3 μmol/g) compared with the controls (9.4 ± 1.2 μmol/g; p = 0.003). The pseudo-first-order CK rate-constant was 26% lower (0.28 ± 0.15 vs 0.38 ± 0.07 s⁻¹, p = 0.035) and the forward CK flux was 44% lower (2.0 ± 1.4 vs 3.6 ± 0.9 μmol/g/s, p = 0.001) than in the controls. The contractile abnormalities did not correlate with the metabolic indexes. In conclusion, myocardial phosphocreatine and CK-ATP delivery are significantly reduced in patients with HC caused by the Arg403Gln mutation, akin to previous results from mice with the same mutation. A lack of a relation between energetic and contractile abnormalities suggests the former result from the sarcomeric mutation and not a late consequence of mechanical dysfunction.

Unilateral isolated hypoglossal nerve palsy associated with internal carotid artery dissection

Genetic linkage and deletion studies have led to the proposal that there are at least two loci on the X chromosome which are responsible for X linked retinitis pigmentosa (XLRP)....

Phenotypic variability of parkin mutations in single kindred

Mutations in parkin have been associated with autosomal recessive early-onset Parkinson's disease (PD). Here, we report on unusual phenotypic variability within a family with mutations in parkin. The proband and her parents were clinically assessed. Mutation analysis was performed using genomic DNA and complementary DNA. The protein expression of Parkin and Mitofusin 2 was examined by western blotting. The proband was a compound heterozygote with no detectable Parkin and presented with early-onset PD. The father, a single heterozygote with reduced expression of Parkin, had mild loss of arm swing. The mother, who had only very mild rigidity, was unexpectedly found to be a homozygote with no Parkin expression. The proband, but not the parents, met the Queen Square Brain Bank criteria for PD. Parkin-dependent ubiquitination of Mitofusin 2 was impaired in the mother and the proband. We report the first case of a homozygous mutation carrier in parkin who had no functional protein and only mild signs of parkinsonism in her seventh decade, whereas her daughter developed typical early-onset PD. This family demonstrates phenotypic variability in parkin-related parkinsonism. © 2012 Movement Disorder Society.