Base Pair Insertion In Exon Research Articles

Somatic hypermutation defects in two adult hyper immunoglobulin M patients

Hyper immunoglobulin M (HIGM) syndrome is a rare disorder of the immune system with impaired antibody functions. The clinical picture of the patients varies according to the underlying genetic variation. In this study, we identified two novel variants in AID and UNG genes, which are associated with autosomal recessive type HIGM, by targeted next-generation sequencing (NGS) panel. A biallelic 11 base pair deletion (c.278_288delATGTGGCCGAC) in the coding sequence of activation-induced cytidine deaminase (AID) gene was identified in a 36-year-old patient. Biallelic two base pair insertion in exon 7 of uracil nucleoside glycosylase (UNG) gene (c.924_925insGG) was identified in a 40-year-old patient. Both variants were confirmed by Sanger sequencing. HIGM, like many of the other primary immunodeficiencies, is a rare and difficult-to-diagnose entity with heterogeneous clinical phenotypes. It should be suspected in patients with a history of early-onset recurrent respiratory infections, enlarged lymph nodes, and autoimmune disorders. There might be a delay in diagnosis until adulthood especially in subtle cases or if HIGM is not included in the differential diagnosis due lacking of awareness. In this regard, genetic testing with NGS-based diagnostic panels provide a rapid and reasonable tool for the molecular diagnosis of patients with immunodeficiencies and hence, decrease the time to diagnose and prevent infection-related complications associated with increased morbidity and mortality.

Role of Kir4.1 (Kcnj10) in the Regulation of Salt‐Induced Hypertension

In the kidney, Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16) are highly expressed in the aldosterone sensitive distal nephron (ASDN), a major target for multiple hormones controlling blood pressure. These basolateral inwardly rectifying potassium channel subunits assemble to form both heteromeric Kir4.1/Kir5.1 channels and homomeric Kir4.1 channels. These channels control the transepithelial voltage, fine‐tune renal electrolyte homeostasis and contribute to long‐term blood pressure control, correspondingly. To study the role of Kir4.1 containing channels in the renal control of blood pressure, renin‐angiotensin‐aldosterone system (RAAS) balance, and salt‐sensitive (SS) hypertension, we created a Kcnj10 knockout (SSKcnj10−/−) on the Dahl SS rat background. The SSKcnj10−/− rat was created using CRISPR/Cas9 mutagenesis, resulting in a single base pair insertion in exon 2 of the Kcnj10 gene and producing a frameshift in Kir4.1 protein translation after only 26 amino acids. Homozygous SSKcnj10−/− rats were hypokalemic, had reduced heart, kidney, and body weights, and did not survive more than 3 weeks after birth. Mass spectrometry based quantification of serum RAAS peptides revealed increased Ang I (1631 vs. 560 pM), Ang 1–5 (499 vs. 69 pM), and Ang 1–7 (598 vs. 42 pM) levels in SSKcnj10−/− rats compared to age‐matched SSKcnj10+/+ controls. No differences were observed in serum Ang II, Ang III, Ang IV, or aldosterone levels. In addition, ACE activity represented by the Ang II to Ang I ratio was decreased in SSKcnj10−/− rats (0.58 vs. 1.1). The increases in alternative RAS axis peptides (Ang 1–5 and Ang 1–7) and decrease in ACE activity in SSKcnj10−/− rats are suggestive of a low blood pressure phenotype, however, their limited survival precluded blood pressure measurement. Heterozygous (SSKcnj10+/−) developed normally and showed no changes in serum electrolytes. Although survival rate of both male and female SSKcnj10+/− rats was dramatically lower compared to SSKcnj10+/+ rats, they were able to survive much longer than SSKcnj10−/− rats (21 vs. 83 days on average, respectively) with dietary K+ supplementation (HK; 1.41% K+), allowing for telemetric blood pressure measurement. We found no differences in baseline blood pressure under control conditions (LS+HK; 123±2 vs 125±4, mmHg), but salt‐induced (HS, 4% NaCl) elevations in blood pressure were significantly attenuated in SSKcnj10+/− rats fed a HS diet (149±6 vs 183±9 mmHg for SSKcnj10+/− and SSKcnj10+/+ rats, respectively). Furthermore, after 3 weeks on HS, SSKcnj10+/− rats showed decreased diuresis (25±5 vs 38±2, ml/day) and improved albuminuria (6.0±4.0 vs 17.2±7.6, Alb/Cre ratio) compared to SSKcnj10+/+ rats. Our results demonstrate that renal Kir4.1 containing channels mediate salt‐sensitive increases in blood pressure through regulation of potassium homeostasis, modulation of alternative RAS axis hormones, and control of renal salt handling in the ASDN.Support or Funding InformationSupported by the National Institutes of Health grants R35 HL135749, R56 DK121750, R24 HL114474, and F31 DK122647

Whole Genome Analysis of a Single Scottish Deerhound Dog Family Provides Independent Corroboration That a SGK3 Coding Variant Leads to Hairlessness.

The breeds of domestic dog, Canis lupus familiaris, display a range of coat types with variation in color, texture, length, curl, and growth pattern. One trait of interest is that of partial or full hairlessness, which is found in a small number of breeds. While the standard for some breeds, such as the Xoloitzcuintli, requires sparse hair on their extremities, others are entirely bald, including the American Hairless Terrier. We identified a small, rare family of Scottish Deerhounds in which coated parents produced a mixed litter of coated and hairless offspring. To identify the underlying variant, we performed whole genome sequencing of the dam and five offspring, comparing single nucleotide polymorphisms and small insertions/deletions against an established catalog of 91 million canine variants. Of 325 homozygous alternative alleles found in both hairless dogs, 56 displayed the expected pattern of segregation and only a single, high impact variant within a coding region was observed: a single base pair insertion in exon two of SGK3 leading to a potential frameshift, thus verifying recently published findings. In addition, we observed that gene expression levels between coated and hairless dogs are similar, suggesting a mechanism other than non-sense mediated decay is responsible for the phenotype.

CRISPR‐Cas9 Gene Editing Yields a Novel Rat Model of Cardiometabolic Disease

Despite strong evidence that the Metabolic Syndrome (MetS) and its defining features (central obesity, dyslipidemia, hypertension, hyperglycemia) are highly heritable, the genetic etiology is complex and relatively few causative genes are known. For this reason, we employ the Lyon Hypertensive (LH) rat, a well‐characterized polygenic inbred MetS model. Previous genetic and computational approaches identified a novel gene (C17h6orf52) as a putative master regulator of gene expression and oxidative phosphorylation with pleiotropic effects on body weight and blood pressure regulation.In this study, two different frameshift mutations were generated independently by CRISPR‐Cas9 gene editing, producing either a 5 base pair deletion or a 10 base pair insertion in exon 2 of C17h6orf52, and females from the resulting mutant rat strains, designated M1 and M2, respectively, were characterized (Table 1). Herein, mutations in C17h6orf52 are shown to significantly increase blood pressure and serum cholesterol in vivo, despite no significant changes in body composition or body weight in LH‐derived female rats.Either mutation appears to cause modest, but significant or nearly significant, increases in blood pressure on a 0.3% NaCl diet. M1−/− females exhibit significantly increased systolic and diastolic blood pressure, while M2−/− females' systolic and diastolic blood pressure is more moderately elevated. This effect is potentiated when animals are administered a high salt diet. Finally, although neither total nor low‐density lipoprotein (LDL) cholesterol is changed in the M1 female rats with respect to their wildtype control, M2 females develop higher serum cholesterol, as well as significantly higher LDL cholesterol.The differences in phenotype between the M1 and M2 mutations were unexpected. Preliminary data indicates that 5 base pair deletion present in the M1−/− rats results in two distinct splicing variants that may be contributing to the observed phenotype differences. Nevertheless, these data suggest that C17h6orf52 is protective against cardiometabolic risk factors, given that C17h6orf52 mRNA is more highly expressed in MetS‐susceptible LH rats than MetS resistant Lyon Normotensive (LN) rats, and the mutation confers a greater burden of MetS features on an already obese and hypertensive rat. The continued study of this rat model of Metabolic Syndrome has the potential to functionally validate an uncharacterized regulatory gene, and provide novel targets for pharmacological intervention in the treatment of obesity.Support or Funding InformationR01HL089895, R21DK089417, T32GM008629, 14GRNT20410043This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Store-operated calcium entry is dispensable for the activation of ERK1/2 pathway in prostate cancer cells

STIM1, the endoplasmic reticulum Ca2+ sensor that modulates the activity of plasma membrane Ca2+ channels, becomes phosphorylated at ERK1/2 target sites during Ca2+ store depletion triggered by thapsigargin or epidermal growth factor (EGF). This ERK1/2-dependent phosphorylation regulates STIM1 localization and dissociation from microtubules, and it is known that enhances the binding to ORAI1, a store-operated Ca2+ entry (SOCE) channel, leading to the activation of this Ca2+ influx pathway. However, there remained some evidence of a role for SOCE in the activation of ERK1/2, and here we assessed the contribution of SOCE to ERK1/2 activation by generating a STIM1-deficient cell line by CRISPR/Cas9 genome editing of the STIM1 locus in prostate cancer PC3 cells. The genomic modification consisted of a 16 base-pair insertion in exon 5 of both alleles, therefore abrogating STIM1 synthesis. STIM1-KO cells did show a striking decrease in Ca2+ influx in response to thapsigargin or EGF, a result that demonstrates that SOCE mediates Ca2+ entry in PC3 cells during stimulation with EGF. Moreover, identical levels of total ERK1/2 were found in STIM1-KO cells and the parental cell line, and ERK1/2 activation was fully activated in KO cells, both in the presence and in the absence of extracellular Ca2+, a result that supports that STIM1 and SOCE are not required for ERK1/2 activation. This activation was sensitive to Src kinase inhibition, but not to CAMKII nor PKC inhibition, a result that sets STIM1 and SOCE as downstream targets of the axis Src-Raf-MEK-ERK, rather than upstream regulators.

Pure Red Cell Aplasia - a New Manifestation of CTLA4 Mutation

The cytotoxic T lymphocyte antigen-4 (CTLA4) is an essential negative regulator of immune responses. Its loss causes fatal autoimmunity in mice. Patients with heterozygous mutations in CTLA4 were recently reported to present with an autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features (Schubert, D. et al., Nat. Med. 20: 1410-1416, 2014; Kuehn, H.S. et al., Science 345, 1623-1627, 2014). Here we report a family with a CTLA4 mutation that presents with hematological manifestations not previously associated with CTLA4 loss.The index case was a girl referred to our hospital with a very severe thrombocytopenia and anemia at the age of 17. Bone marrow examination revealed no megakaryocytes and just a few erythroblasts. In contrast, granulopoiesis was normal and clonal lymphocytes were not detected. No etiology of the lack of thrombopoiesis and erythropoiesis was discovered. Lack of response to high-dose steroid treatment, platelet counts below 10x109/l, short-lasting effect of transfused platelets and a bleeding tendency urged us to start treatment with horse anti-thymocyte globulin (ATG) and cyclosporine after 2 months. A positive clinical response was observed in 2 weeks. Hb and platelet counts were normalized by 4 weeks, and all medication discontinued by 5 months. Two months later, the patient suffered from generalized tonic-clonic seizures. MRI revealed multiple cerebral lesions, lymphadenopathy and multiple infiltrates in lungs and kidneys. A core kidney biopsy revealed no clonal lymphoid infiltrates. A cerebral biopsy suggested crystal-storing histiocytosis. Steroids were administered, and the infiltrates disappeared gradually during the following months. More than two years later the patient suffered from a Salmonella-induced gastroenteritis, and again she became severely anemic. Her bone marrow revealed now lack of erythropoiesis whereas thrombopoiesis and granulopoiesis were normal. She was then diagnosed with pure red cell aplasia (PRCA).An uncle of the index case had been referred to us with the diagnosis MDS at the age of 47, one year prior to the index patient's last referral and PRCA diagnosis. Dysplasia was not verified, and since the patient completely lacked red cell precursors, the diagnosis was changed to PRCA. In addition, both he, his mother and the index case had been suffering from diarrhea for years without any diagnosis. His mother also had pernicious anemia.The two familial cases of PRCA, a rare hematological manifestation, prompted us to conduct whole exome sequencing which revealed that the two patients with PRCA were both heterozygous for a 5 base pair-insertion in exon 1 of the CTLA4 gene, leading to a frameshift and premature stop codon causing haploinsufficiency. Familial segregation was tested by Sanger sequencing and showed that also the grandmother and the mother of the index case were carriers of the same mutation.Functional assays of our index patient, the two family members with autoimmunity (her uncle and grandmother) and her unaffected mother revealed fewer CD19+ B- and naïve T cells than normal. Furthermore, we found dysregulation of regulatory T (Treg) cells, reduced levels of T cell CTLA4, particularly FoxP3+ Treg CTLA4 (mean fluorescence intensity as well as frequency of CTLA4+ T cells and Tregs) and signs of hyperactivity of effector T cells. Our findings were as reported in the above cited publications.The discovery of a CTLA4 mutation in this family with 3 affected members and one unaffected carrier might have immediate implications for our index patient. As she did not respond to high-dose steroids as her uncle did, her genetic diagnosis opens up to additional treatment opportunities. Actual medication for patients with mutations in CTLA4 and autoimmunity could be mTOR inhibitors like sirolimus and possibly CTLA4 fusion proteins (abatacept and belatacept). These fusion proteins bind to CD80 and CD86 and inhibit CD28-mediated immune activation.In conclusion, we have detected a mutation in the CTLA4 gene with corresponding T cell aberrations in this family through the new manifestation PRCA. These findings may be of clinical importance for the family we have characterized as well as for other patients with similar manifestations. DisclosuresOff Label Use: Abatacept and belatacept are fusion protein consisting of a part of CTLA4. It can modulate a signal necessary for activating specific T lymphocytes and can be effective in patients with CTLA4 mutations..

Two novel RUNX1 mutations in a patient with congenital thrombocytopenia that evolved into a high grade myelodysplastic syndrome

Here we report two new RUNX1 mutations in one patient with congenital thrombocytopenia that transformed into a high grade myelodysplastic syndrome with myelomonocytic features. The first mutation was a nucleotide base substitution from guanine to adenine within exon 8, resulting in a nonsense mutation in the DNA-binding inhibitory domain of the Runx1 protein. This nonsense mutation is suspected a de novo germline mutation since both parents are negative for the mutation. The second mutation identified was an in-frame six nucleotide base pair insertion in exon 5 of the RUNX1 gene, which is predicted to result in an insertion in the DNA-binding runt homology domain (RHD). This mutation is believed to be a somatic mutation as it was mosaic before allogeneic hematopoietic cell transplantation and disappeared after transplant. As no other genetic mutation was found using genetic screening, it is speculated that the combined effect of these two RUNX1 mutations may have exerted a stronger dominant negative effect than either RUNX1 mutation alone, thus leading to a myeloid malignancy.

Advances of intravenous immunoglobulin G in modulation of anti-fetal immunity in selected at-risk populations: science and therapeutics.

Advances of intravenous immunoglobulin G in modulation of anti-fetal immunity in selected at-risk populations: science and therapeutics.

Sensitive methods for the detection of an insertion in exon 20 of the HER2 gene in the metastasis of non-small cell lung cancer to the central nervous system

The HER2 (ErbB2/neu) protein is a member of the HER (ErbB) receptor family (EGFR, HER2, HER3 and HER4) that expresses tyrosine kinase activity in the intracellular domain. EGFR and HER2 overexpression is observed in numerous types of cancer, nevertheless, the susceptibility of patients with non-small cell lung cancer (NSCLC) to therapy with EGFR and HER2 tyrosine kinase inhibitors (TKIs) depends on mutations present in the respective coding genes (driver mutations). In the present study, PCR and amplified DNA fragment length analysis (FLA) were used along with the multi-temperature single-strand conformation polymorphism (MSSCP) technique in order to identify the 12 base pair insertion in exon 20 of the HER2 gene in 143 patients with NSCLC metastasis to the central nervous system. The prevalence of the HER2 gene mutation was correlated with mutations in the EGFR and BRAF genes. The insertion in exon 20 of the HER2 gene was observed in a single 77-year-old, non-smoking male, with poorly-differentiated adenocarcinoma of the lung (1.5% of adenocarcinoma patients). No other genetic abnormalities were identified in this patient. In the therapy of NSCLC patients with HER2 gene mutations, drugs that inhibit the EGFR and HER2 receptors, for example afatinib, may be effective. The identification of other driving mutations in NSCLC cells appears to be key to the appropriate qualification of molecular targeted therapies.

Novel mutations in the MNX1 gene in two families with Currarino syndrome and variable phenotype

The Currarino syndrome (CS) consists of a sacral defect, an anorectal malformation and a pre-sacral mass. It manifests as an autosomal dominant congenital malformation in familial settings, with varying penetrance. The disease-causing gene, Motor neuron and pancreas homeobox-1 (MNX1), is known to be mutated in almost all familial cases, but due to the lack of genotype–phenotype correlation, there is a need for better clinical and molecular genetic characterization of the CS. Here, we report two novel mutations in the MNX1 gene in two cases. Each case was found to be familial upon further investigation of the other members of each family. The first affected case (a one year old boy) exhibited a missense mutation, p.Phe289Ser, in exon 3 in the highly conserved third helix of the homeodomain, which is considered to affect the DNA binding property and transcription regulation of the protein. The mutation seemed to display full penetrance of the disease in this family, but with different time of on-set. The second affected case (a 5months old boy) displayed a 13 basepair insertion in exon 1, creating a complex frameshift mutation which results in a premature truncation of the protein that lacks the third helix homeodomain. Other members of the boy's family, who harbored the same mutation, were found to be completely asymptomatic. In conclusion, we detected two novel mutations in the MNX1 gene in cases with CS, which supports mutational analysis in the diagnosis of CS, even though the variability in the genotype and phenotype correlation maintains.

Maternal homozygocity for a 14 base pair insertion in exon 8 of the HLA-G gene and carriage of HLA class II alleles restricting HY immunity predispose to unexplained secondary recurrent miscarriage and low birth weight in children born to these patients

Homozygous carriage of a 14 base pair (bp) insertion in exon 8 of the HLA-G gene may be associated with low levels of soluble HLA-G and recurrent miscarriage (RM). We investigated the G14bp insertion(ins)/deletion(del) polymorphism in 339 women with unexplained RM and 125 control women. In all patients and patients with secondary RM after a firstborn boy, 19.2% and 23.9%, respectively, were G14bp ins/ins compared with 11.2% of controls (p<0.05 and p<0.01). Among secondary RM patients with a firstborn boy, G14bp del/del and no carriage of an HLA class II (HYrHLA) allele restricting immunity against male-specific minor HY antigens was found less often than in controls (p<0.05) whereas G14bp ins/ins and carriage of HYrHLA predisposed (p<0.08) to this clinical entity. The mean birth weight of firstborn boys born to G14bp ins positive secondary RM patients was significantly lower than expected (p<0.001) but only in carriers of HYrHLA alleles (p<0.01). In conclusion, homozygosity for G14bp ins predisposes to RM. The combination of G14 ins homozygosity and carriage of HYrHLA predisposes to secondary RM in women with a firstborn boy and negatively affects birth weight in these boys.

A 14 basepair insertion in exon 8 of the maternal HLA-G gene is associated with low soluble HLA-G concentrations during pregnancy

A 14 basepair insertion in exon 8 of the maternal HLA-G gene is associated with low soluble HLA-G concentrations during pregnancy

Novel LRP5 gene mutation in a patient with osteoporosis-pseudoglioma syndrome

Osteoporosis-pseudoglioma syndrome (OPPG) is a rare autosomal recessive disorder characterised by severe juvenile-onset osteoporosis and congenital or early-onset blindness. This serious illness is due to mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) that is a major actor in pathways involved in bone remodelling. Here, we report a novel frameshift mutation identified in a 22 year-old Tunisian boy of a consanguineous family. This patient had low bone mineral density (BMD), experienced multiple fractures during childhood and suffered ocular alterations with blindness. Direct DNA sequencing showed a homozygous 5 base pair insertion in exon 5 of the LRP5 gene. This new mutation is located in the first EGF-like domain and gives rise to a truncated protein of 384 amino acids. The functional significance of this mutation clearly indicates a loss-of-function mutation of the LRP5 gene leading to the observed OPPG phenotype. Rheumatologists must be aware of LRP5 gene that in addition to being a major gene in the mendelian disease that is OPPG syndrome seems to be involved in osteoporosis in the general population through some of its polymorphisms.

Preimplantation genetic diagnosis of P450 oxidoreductase deficiency and Huntington Disease using three different molecular approaches simultaneously

Description of the confluence of different molecular techniques to detect three different mutations in one cell. The man carries a 20 base pair insertion in exon 12 of the POR gene (c.1551_1552ins20), and the woman carries a point mutation in exon 8 of the POR gene (c.859G>C) plus a triplet repeat expansion in the HTT gene. Huntington Disease (HD) had to be diagnosed using short tandem repeat (STR) markers linked to the HTT gene. The mutation c.1551_1552ins20 was analyzed by fragment size and c.859G>C was minisequenced. Furthermore, STR markers linked to the POR gene were included to support the diagnosis of P450 oxidoreductase (POR) deficiency. Nine embryos were diagnosed in total: three as POR deficiency affected, two as HD affected, one as POR deficiency and HD affected, and two as carriers of the paternal POR deficiency mutation and healthy for HD. These two last embryos were transferred but no pregnancy was achieved. A successful procedure combining direct and indirect methods for the detection of three different mutations in a single cell has been achieved for the first time.

A four base pair insertion in exon 1 of the RPS19 gene is a common polymorphism in African‐Americans

A four base pair insertion in exon 1 of the <i>RPS19</i> gene is a common polymorphism in African‐Americans

Deletion of 17p13 and LIS1 Gene Mutation in Isolated Lissencephaly Sequence

Classical lissencephaly is a neuroblast migration disorder that occurs either as isolated lissencephaly sequence or in association with malformation syndromes, such as the Miller-Dieker syndrome. In this work, alterations of the LIS1 gene in patients diagnosed as having isolated lissencephaly sequence were investigated. Ten patients were evaluated for the following aspects: classical cytogenetics by karyotyping using solid staining and G-banding; molecular cytogenetics using fluorescent in situ hybridization with a specific probe for the critical region of isolated lissencephaly sequence; and molecular analysis using deoxyribonucleic acid sequencing. Classical cytogenetic analysis indicated apparently normal karyotypes in all patients, but fluorescent in situ hybridization revealed a 17p13.3 microdeletion in one. In another patient, deoxyribonucleic acid sequencing disclosed a 1 base pair insertion in exon 4 within a sequence of eight consecutive adenine residues (162-163insA), a mutation that predicts a truncated protein. Two different polymorphisms were also detected: a T>C substitution in intron 6 (c.568 + 27bp T>C) and a C>T substitution in the nontranslated region of exon 11 (1250 C>T). These results indicate that cytogenetic analysis and molecular investigation of the LIS1 gene are not always sufficient to determine the disease etiology. These findings are consistent with previous studies and suggest the involvement of other genes in cortical malformation.

Mutation analysis of NR2E3 and NRL genes in Enhanced S Cone Syndrome.

Ten new and seventeen previously reported Enhanced S Cone Syndrome (ESCS) subjects were used to search for genetic heterogeneity. All subjects were diagnosed with ESCS on the basis of clinical, psychophysical and/or electroretinography testing using published criteria. Mutation analysis was performed on the NR2E3 nuclear receptor gene by single strand conformation analysis and direct sequencing, which revealed either homozygous (N=13) or compound heterozygous (N=11) mutations in 24 subjects (89%), heterozygous mutations in 2 subjects (7%) and no mutations in 1 subject (4%). Fifteen different mutations were identified, including six not previously reported. The subject (Patient A) with no detected NR2E3 mutation had features not usually associated with ESCS, in particular moderate rod photoreceptor function in peripheral retina and an abnormally thick retinal nerve fibre layer. Mutation analysis of the NRL, CRX, NR1D1 and THRB genes in this individual revealed a heterozygous one base-pair insertion in exon 2 of the NRL gene, which results in a predicted truncation of the NRL protein. Loss-of-function NRL alleles have not been described previously in humans, but since the same mutation was present in unaffected family members, it raises the possibility that the abnormal ESCS phenotype in Patient A may result from a digenic mechanism, with a heterozygous NRL mutation and a mutation in another unknown gene.

Autosomal recessive hypercholesterolemia: genetics and clinical aspects

In the last 10 years, new types of genetic hypercholesterolemia, different from familial hypercholesterolemia (FH) or familial defective apo B (FDB), have been reported; these forms have both autosomal dominant or recessive inheritance. Starting in 1995, we have described a new recessive type of hypercholesterolemia, named ARH. This disease, initially reported in one Sardinian family, is characterized by a severe increase of low-density lipoproteins (LDL) in plasma, xanthomas, xanthelasmas, and premature coronary heart disease. The family showed presence of consanguinity, bimodal distribution of cholesterol levels, and absence of vertical transmission. We have demonstrated that this hypercholesterolemia is due to a marked reduction of LDL catabolism that is caused by a selective reduction of LDLs uptake by the liver. The gene responsible for ARH maps to chromosome 1p35. It codes for an adaptor protein that interacts with the NPXY sequence in the cytoplasmic tail of LDL-R, and is necessary for the functioning of LDL-R in liver, but not in fibroblasts. Only two mutations were identified in Sardinia; a single base pair insertion in exon 4 (ARH1), and a nonsense mutation at codon 22 (ARH2). The high frequency of ARH in the island is probably due to a combination of genetic drift, consanguinity, and geographic isolation.

Mutations in the ganglioside-induced differentiation-associated protein-1 (GDAP1) gene in intermediate type autosomal recessive Charcot-Marie-Tooth neuropathy.

Mutations in the gene for the ganglioside-induced differentiation-associated protein-1 (GDAP1) on 8q21 recently were reported to cause autosomal recessive Charcot-Marie-Tooth (CMT) sensorimotor neuropathy. Neurophysiology and nerve pathology were heterogeneous in these cases: a subset of GDAP1 mutations was associated with peripheral nerve demyelination, whereas others resulted in axonal degeneration. In this study, we identified two novel mutations disrupting the GDAP1 reading frame. Homozygosity for a single base pair insertion in exon 3 (c.349_350insT) was observed in affected children from a Turkish inbred pedigree. The other novel allele detected in a German patient was a homozygous mutation of the intron 4 donor splice site (c.579 + 1G>A). Patients with GDAP1 mutations displayed severe, early childhood-onset CMT neuropathy with prominent pes equinovarus deformity and impairment of hand muscles. Nerve conduction velocities were between 25 and 35 m/s and peripheral nerve pathology showed axonal as well as demyelinating changes. These findings fitted the definition of intermediate type CMT and further support the view that GDAP1 is vital for both, axonal integrity and Schwann cell properties.

Characterization of non-expressed C4 genes in a case of complete C4 deficiency: identification of a novel point mutation leading to a premature stop codon

The genetic basis of complete C4 deficiency in a patient with SLE was investigated. Previous studies have demonstrated that this patient has two different major histocompatibility complex (MHC) haplotypes that each contain a major deletion and a non-expressed C4 gene. In the present study, non-expression of the C4 genes was explained by the finding of two distinct C4 gene mutations. A previously described two base pair insertion in exon 29 of the C4 gene was detected in the paternal MHC haplotype [HLA-A2, B40, SC00, DR6]. The maternal haplotype [HLA-A30, B18, F1C00, DR3] carried a C4 gene with a one base pair deletion in exon 20 generating a premature stop codon. This mutation was neither found in 10 individuals with known non-expressed C4 genes nor in 9 individuals homozygous for the complotype F1C30. The isotype and allotype specific regions of the patient’s C4 genes were sequenced, and both contained C4A3a sequence. In conclusion, two different MHC haplotypes resembling the extended haplotypes [HLA-A2, B40, SC02, DR6] and [HLA-A30, B18, F1C30, DR3] both contained a non-expressed C4A gene that was due to either of two distinct mutations, demonstrating the heterogeneous genetic background of C4 deficiency.