Family-based genetic analysis in schizophrenia by whole-exome sequence to identify rare pathogenic variants.

Schizophrenia (SCZ) is influenced by a combination of genetic and environmental factors. Although several studies have been conducted to identify the causative loci and genes, few of these loci or genes can be repeated due to the high phenotypic and genetic heterogeneity of disease, and their mechanisms are not fully understood. There may be some "missing heritability" that has not yet been found. In order to investigate the deleterious heritable mutations, whole-exome sequencing (WES) in pedigrees with SCZ was used in the current work. Two unrelated pedigrees with SCZ were recruited to perform WES. Genetic analysis was next performed to find potential variants in accordance with the prioritized strategy. Followed by genetic analysis to detect candidate variants according to the prioritized strategy. Next, a series of algorithms was used to predict the pathogenicity of variants. Sanger sequencing was finally conducted to verify the co-segregation. Recessive mutations in six genes (TFEB, SNAI2, TFAP2B, PRKDC, ST18 in Pedigree 1 and PKHD1L1 in Pedigree 2) that co-segregated with SCZ in two families were discovered through genetic analysis by WES. Sanger sequencing verified that all of the mutations in the affected siblings were homozygous. These results corroborated the hypothesis that SCZ exhibits strong heterogeneity and complex inheritance patterns. The newly discovered homozygous variations deepen our understanding of the mutation spectrum and offer more proof for the involvement of TFEB, SNAI2, TFAP2B, PRKDC, ST18, and PKHD1L1 in the development of SCZ.

Mitochondrial uncoupling protein 2 (UCP2) gene polymorphism - 866 G/A in the promoter region is associated with type 2 diabetes mellitus among Kashmiri population of Northern India.

OBJECTIVE: The study aimed to evaluate the association of UCP2 gene polymorphism - 866 G/A and its expression with diabetes predisposition in the North Indian population. METHODS: The study involved 850 subjects, including 425 each T2DM and control subjects. The serum metabolic and clinical parameters were estimated using standard protocols. The PCR-RFLP based genotyping was performed to determine UCP2 gene polymorphism, while the expression was measured by real-time quantitative PCR. RESULTS: The genotypic and allelic frequencies showed a significant difference in cases compared to controls (p < 0.05). The diabetes patients had a 4.2-fold decrease in UCP2 gene expression. The expression was 29.8 and 8.4 fold lower in diabetes patients with homozygous (AA) and heterozygous (GA) mutation at - 866 locus of UCP2 nucleotide sequence, respectively. When categorized according to age and BMI, the T2DM subjects with age ≥ 50 and BMI ≥ 25 had a 5.53 and 8.2-fold decrease in UCP2 expression, respectively. The diabetes subjects with homozygous and heterozygous mutation demonstrated a pathological increase in serum metabolic and clinical parameters, which corroborated with UCP2 gene expression, indicating a strong association between the two. Intriguingly, we did not find any association between - 866 G/A polymorphism of UCP2 with serum insulin levels. CONCLUSION: Our investigation is the first among the studies conducted in Jammu and Kashmir to work on adipose tissue and UCP2 gene polymorphism. The association of - 866 G/A SNP of the UCP2 gene with its expression in diabetes patients appears to be an important genetic determinant in the progression of T2DM. Moreover, age ≥ 50 years and BMI ≥ 25 could be considered risk factors for developing T2DM in the studied population.

Disease severity and genotype-phenotype correlation in adult patients with familial Mediterranean fever.

OBJECTIVES: To assess the relationships of disease severity with genotype and phenotype in adult familial Mediterranean fever patients. METHODS: Two-hundred seventy-five patients included in the study were divided into four groups according to their mutations: Group 1, M694V homozygous; Group 2, M694V-other; Group 3, other-other; and Group 4, no meaningful gene variants. Disease severity was evaluated using the Pras disease severity score. The association between Pras scores and other possible predictors was assessed by the multiple linear regression analysis. RESULTS: In this study, 12.4% of all patients were in Group 1, 55.3% were in Group 2, 26.5% were in Group 3, and 5.8% were in Group 4. Pras scores were higher in Group 1 than in Groups 2, 3, and 4 (post hoc pairwise comparisons; P = .001, P < .001, and P = .001, respectively). Age at disease onset and age at diagnosis were found moderately and strongly correlated with Pras scores. Patients with moderate and severe disease were intensely involved in Group 1. CONCLUSIONS: Higher Pras scores, earlier age of symptoms and diagnosis, more frequent arthritis and erysipelas-like erythema, and higher colchicine dose are closely associated with M694V homozygous familial Mediterranean fever patients. These patients also have mostly moderate and severe disease severity.

Autosomal recessive cutis laxa type 1C with a homozygous LTBP4 splicing variant: a case report and update of literature.

BACKGROUND: Autosomal recessive cutis laxa (ARCL) is a heterogeneous disorder with three primary forms (ARCL 1, ARCL 2 and ARCL 3). Latent transforming growth factor beta binding protein 4 (LTBP4) anomalies cause ARCL1C and are connected to different problems in the skin and other organs. Herein, we present a seven month old Iranian boy with a clinical manifestation of ARCL1 with literature review of previous cases with attributes of ARCL1C. METHODS: Considering the craniofacial characteristics and respiratory distress of the proband, cutis laxa (CL) was expected and whole-exome sequencing (WES) was performed. RESULTS: In the proband, signs of CL were mainly located in the face, thorax, and abdomen. The prenatal investigation revealed a diaphragmatic hernia and certain uncommon signs, such as an atrial septal defect and pyloric stenosis. The WES showed a novel homozygous mutation (c.533-1G > A) in exon six of the LTBP4 gene. CONCLUSION: This report showed a new variant with uncommon clinical features, such as a stenosis atrial septal defect and pyloric stenosis, which causes ARCL1C. Unfortunately, the proband developed several heart problems and died at the age of seven months and seven days. Thus, a more in-depth evaluation is needed to clarify the different aspects of CL related to LTBP4 disorder.

Moyamoya Disease Associated with a Deficiency of Complement Component 6.

OBJECTIVES: Complement component 6 (C6) deficiency is a very rare genetic defect that leads to significantly diminished synthesis, secretion, or function of C6. In the current report, we demonstrate a previously undescribed, homozygous missense mutation in exon 17 of the C6 gene (c.2545A>G p.Arg849Gly) in a 35-year-old Japanese woman with moyamoya disease and extremely low levels of CH50 (<7.0 U/mL). MATERIALS AND METHODS: The complement gene analysis using hybridization capture-based next generation sequencing was performed. CH50 was determined in patient's plasma mixed with plasma from a healthy donor or purified human C6 protein. Western blot was performed on patient's plasma using polyclonal antibodies against C6, with healthy donor's plasma and purified human C6 protein as positive controls while C6-depleted human serum as a negative control. The carriage of ring finger protein 213 variant (c.14576G>A p.Arg4859Lys), a susceptibility gene for moyamoya disease, was examined by direct sequencing. RESULTS: CH50 mixing test clearly showed a deficiency pattern, being rescued by addition of only 1% healthy donor's plasma or 1 µg/mL purified human C6 protein (1/50-1/100 of physiological concentration). Western blot revealed the absence of C6 protein in the patient's plasma, confirming a quantitative deficiency of C6. The ring finger protein 213 variant was not detected. CONCLUSIONS: Our data implies that unrecognized complement deficiencies would be harbored in cerebrovascular diseases with unknown etiologies.

Genomic analysis of a riboflavin-overproducing Ashbya gossypii mutant isolated by disparity mutagenesis.

BACKGROUND: Ashbya gossypii naturally overproduces riboflavin and has been utilized for industrial riboflavin production. To improve riboflavin production, various approaches have been developed. In this study, to investigate the change in metabolism of a riboflavin-overproducing mutant, namely, the W122032 strain (MT strain) that was isolated by disparity mutagenesis, genomic analysis was carried out. RESULTS: In the genomic analysis, 33 homozygous and 1377 heterozygous mutations in the coding sequences of the genome of MT strain were detected. Among these heterozygous mutations, the proportion of mutated reads in each gene was different, ranging from 21 to 75%. These results suggest that the MT strain may contain multiple nuclei containing different mutations. We tried to isolate haploid spores from the MT strain to prove its ploidy, but this strain did not sporulate under the conditions tested. Heterozygous mutations detected in genes which are important for sporulation likely contribute to the sporulation deficiency of the MT strain. Homozygous and heterozygous mutations were found in genes encoding enzymes involved in amino acid metabolism, the TCA cycle, purine and pyrimidine nucleotide metabolism and the DNA mismatch repair system. One homozygous mutation in AgILV2 gene encoding acetohydroxyacid synthase, which is also a flavoprotein in mitochondria, was found. Gene ontology (GO) enrichment analysis showed heterozygous mutations in all 22 DNA helicase genes and genes involved in oxidation-reduction process. CONCLUSION: This study suggests that oxidative stress and the aging of cells were involved in the riboflavin over-production in A. gossypii riboflavin over-producing mutant and provides new insights into riboflavin production in A. gossypii and the usefulness of disparity mutagenesis for the creation of new types of mutants for metabolic engineering.

E1021K Homozygous Mutation in PIK3CD Leads to Activated PI3K-Delta Syndrome 1.

PURPOSE: Activated PI3Kδ syndrome 1 is a primary immunodeficiency disease, usually caused by heterozygous mutations in PIK3CD. We aimed to identify the cause of homozygous mutation at c.G3061A (p.E1021K) in a patient and the effect of allele dose in this mutation. METHODS: Genomic DNA from the parent-child trio was analyzed by next-generation sequencing. We performed phenotypic analyses in the patient and in Pik3cdE1024K+/+ mice. RESULTS: The patient was a girl harboring a homozygous mutation for p.E1021K in PIK3CD. At the age of 2 months, she began experiencing respiratory tract infections and lymphoproliferation, accompanied by bronchiectasis and extensive atelectasis in the lungs. She suffered from Haemophilus influenzae and Cytomegalovirus infections and experienced restricted growth and development. Whole-exome sequencing showed a region that included PIK3CD, with loss of heterozygosity (LOH) in chromosome 1 of the patient. The patient had not inherited any allele from her father in the LOH region. Copy number variation analysis showed no changes in the patient's father and the patient. Ultra-deep sequencing of genomic DNA from the patient's mother showed that the mutant allele frequency for c.G3061A was 1.64%. Thus, the presence of segmental maternal uniparental disomy and maternal gonosomal mosaicism resulted in the homozygous mutation. Lymphadenopathy, differentiation of activated T cells, and follicular B cells lymphopenia were found to be more prominent in Pik3cdE1024+/+ mice than in Pik3cdE1024+/- mice. CONCLUSION: This report showed the coexistence of uniparental disomy and mosaicism in PIK3CD. Some immunological features were seen to be allele dose-dependent in the presence of p.E1021K mutation.

A novel homozygous mutation p.E88K in maternal SLC30A2 gene as a cause of transient neonatal zinc deficiency.

The SLC30A2 gene encodes zinc transporter ZnT2, which is indispensable for the transport of zinc into the breast milk in the mammary gland. Transient neonatal zinc deficiency (TNZD) is caused by a mutation in the maternal SLC30A2 gene and has a clinical presentation similar to that of acrodermatitis enteropathica (AE). We described the case of a Chinese infant who presented with AE-like lesions 10 days after birth. Sanger sequencing of the AE-causing gene SLC39A4 revealed no mutations in genomic DNA from the infant, excluding the possibility of AE. Detection of the mother's breast milk showed a significantly lower zinc level. Thus, SLC30A2 sequencing was performed on her genomic DNA and a previously unreported homozygous c.262G > A (p.E88K) mutation was disclosed. Functional analysis suggested the novel mutation could lead to a strong disruption of zinc secretion, which indicated a complete loss of function in the ZnT2 protein. We finally diagnosed the infant with TNZD. To the best of our knowledge, this is the first case of TNZD caused by a homozygous mutation in the maternal SLC30A2 gene. Compared to the heterozygous condition, a homozygous mutation seems to result in a more significant decrease in zinc secretion and a more rapid onset of TNZD.

PPP1R21-related syndromic intellectual disability: Report of an adult patient and review.

Variants in PPP1R21 were recently found to be associated with an autosomal recessive intellectual disability syndrome in 9 individuals. Our patient, the oldest among the known subjects affected by PPP1R21-related syndrome, manifested intellectual disability, short stature, congenital ataxia with cerebellar vermis hypoplasia, generalized hypertrichosis, ulcerative keratitis, muscle weakness, progressive coarse appearance, macroglossia with fissured tongue, and deep palmar and plantar creases. We provide an overview of the clinical spectrum and natural history of this newly recognized disorder, arguing the emerging notion that PPP1R21 gene mutations could result in endolysosomal functional defects. The oldest patients could display a more severe clinical outcome, due to accumulation of metabolites or damage secondary to an alteration of the autophagy pathway. Follow-up of patients with PPP1R21 mutations is recommended for improving the understanding of PPP1R21-related syndromic intellectual disability.

A homozygous MITF mutation leads to familial Waardenburg syndrome type 4.

Waardenburg syndrome (WS) is a genetic disorder characterized by hearing loss and pigmentary abnormalities with variable penetrance. Though heterozygous mutations in MITF are a major cause for Waardenburg syndrome type 2 (WS2), homozygous mutations in this gene and the associated phenotype have been rarely characterized. In this study, we identified a novel p.R223H mutation in MITF in a Chinese Han family with variable WS features. Both parents carried a heterozygous p.R223H mutation. They had normal hearing, and premature greying of the hair is their only pigmentary abnormality. In contrast, their two children both carried a homozygous p.R223H mutation and had classic WS features including profound hearing loss, heterochromia irides and marked pigmentary abnormalities in hair and skin. Interestingly, the two affected children also have persistent chronic constipation since the neonatal period, symptoms suggestive of Waardenburg syndrome type 4 (WS4). Our study revealed a likely association between homozygous mutations in MITF and WS4, which implies a dosage effect for the underlying pathogenesis mechanism.

Skin-Dominant Phenotype in a Patient with H Syndrome: Identification of a Novel Mutation in the SLC29A3 Gene.

H syndrome (OMIM 602782) is a very rare autosomal recessive genodermatosis with multisystem involvement. Hallmarks of this disorder are juvenile onset and progressive, hyperpigmented, hypertrichotic lesions with histiocytic infiltration. Associated systemic manifestations form a long list, and there is high variability between patients. In some patients, dysmorphic and other systemic features may be so subtle that the disorder may readily be mistaken as an acquired skin disease and treated as such. Herein, we report a novel homozygous c.1339G>A (p.Glu447Lys) mutation in the SLC29A3 gene in a patient with skin-dominant presentation of H syndrome. Additionally, due to the present case, double superior vena cava can be added to the list of possible cardiovascular manifestations of H syndrome.

Confirming the recessive inheritance of SCN1B mutations in developmental epileptic encephalopathy.

Dominant SCN1B mutations are known to cause several epilepsy syndromes in humans. Only 2 epilepsy patients to date have been reported to have recessive mutations in SCN1B as the likely cause of their phenotype. Here, we confirm the recessive inheritance of 2 novel SCN1B mutations in 5 children from 3 families with developmental epileptic encephalopathy. The recessive inheritance and early death in these patients is consistent with the Dravet-like phenotype observed in Scn1b-/- mice. The 'negative' clinical exome in one of these families highlights the need to consider recessive mutations in the interpretation of variants in typically dominant genes.

A novel homozygous UMOD mutation reveals gene dosage effects on uromodulin processing and urinary excretion.

Heterozygous mutations in UMOD encoding the urinary protein uromodulin are the most common genetic cause of autosomal dominant tubulointerstitial kidney disease (ADTKD). We describe the exceptional case of a patient from a consanguineous family carrying a novel homozygous UMOD mutation (p.C120Y) affecting a conserved cysteine residue within the EGF-like domain III of uromodulin. Comparison of heterozygote and homozygote mutation carriers revealed a gene dosage effect with unprecedented low levels of uromodulin and aberrant uromodulin fragments in the urine of the homozygote proband. Despite an amplified biological effect of the homozygote mutation, the proband did not show a strikingly more severe clinical evolution nor was the near absence of urinary uromodulin associated with urinary tract infections or kidney stones.

A Turkish case of galactosialidosis with a new homozygous mutation in CTSA gene.

Galactosialidosis is an autosamal reressive lysosomal storage disease caused by a combined deficiency of lysosomal ß-galactosidase and neuraminidase, due to a primary defect in protective protein/cathepsin A. Three subtypes are recognized: the early infantile type, the late infantile type, and the juvenile/adult type. We report here a female patient with early infantile galactosialidosis who was born at 35 weeks of gestation. After birth she remained at the neonatal intensive care unit. Physical examination revealed, coarse facial features, hepatomegaly, cardiac murmur and diffuse hypotonia. The patient's mother had a past history of fetal hydrops history. The diagnosis of galactosialidosis was confirmed by decreased activity of ß-galactosidase and undetectable neuraminidase activity in fibroblasts. Genetic examination revealed a new homozygous mutation (c.1284delG) in the CTSA gene.

Increase in NRAS mutant allele percentage during metastatic melanoma progression.

One-fifth of cutaneous melanomas have dominant gain-of-function mutations of the NRAS oncogene. We report the first two cases of increasing NRAS mutant allele frequency in melanoma metastases and show that the chromosomal mechanism of this homozygosity is an increased polysomy of chromosome 1. We observed an increase in NRAS mutant allele percentage (NRAS-MA%) in the metastatic melanoma progression from 2 patients with melanomas harbouring a NRAS mutation (p.Q61K in case 1 and p.Q61R in case 2). In case 1, we observed a NRAS-MA% increase from 18% within the first metastatic node to 81%, 92% and 85% respectively in the three subsequent metastases: lymph node, brain and subcutaneous metastases biopsied 1, 6 and 17 months, respectively, after the initial lymph node biopsy. In case 2, we observed an increase in NRAS-MA% from 40% within the primary melanoma to 63% within the metastatic lymph node. FISH analysis showed the same results in both cases: a frequent polysomy of chromosome 1 in metastasis samples with NRAS mutant allele percentage >60%, while most cells were disomic in the samples with well-balanced heterozygous mutations. The percentage of NRAS mutant allele may increase during metastatic progression and may be associated with chromosomal instability. Further studies are needed to evaluate the prognostic impact of the NRAS homozygous status and/or polyploidy in metastatic cutaneous melanomas.

A novel SLC12A3 gene homozygous mutation of Gitelman syndrome in an Asian pedigree and literature review.

OBJECTIVES: Gitelman syndrome (GS) is an autosomal recessive disease characterized by hypokalemic metabolic alkalosis in combination with significant hypomagnesemia and hypocalciuria which is caused by mutations in the SLC12A3 gene. In this study, we reported a case of GS pedigree and reviewed pertinent literature so as to explore the relationship between clinical characteristics and genotype meanwhile provide recommendations for the diagnosis and treatment of GS. DESIGN AND METHODS: This is a pedigree-based genetic study of GS and 11 members from one family were included. We summarized their clinical features, analyzed laboratory parameters related to GS and SLC12A3 gene. RESULTS: The proband experienced intermittent severe symptoms of weakness accompanied by significant hypokalemia, hypomagnesemia and hypocalciuria in laboratory test with poor treatments. His mother had more slight symptoms of weakness than him with mild hypokalemia and hypocalciuria. Mild hypomagnesemia was also observed in his sister with occasional weakness. All other pedigree members had normal laboratory test with no GS-related symptoms. A homozygous mutation of SLC12A3 gene (c.488C > T) was detected by genetic testing in three members, and six were carriers of this mutation. CONCLUSIONS: Genotype and phenotype vary significantly among GS patients. Male patients tend to experience more severe symptoms and poor treatment effect. Further large-scale population, animal, and molecular biology experiments are required to investigate the complexity of GS and to find a better treatment regimen for this disease.

Role of Leptin Deficiency, Inefficiency, and Leptin Receptors in Obesity.

Leptin protein consists of 167 amino acids, which is mainly secreted from the white adipose tissue. This protein acts on the hypothalamic regions of the brain which control eating behavior, thus playing a significant role in maintaining body's metabolism. Leptin receptors belong to glycoprotein 130 (gp130) family of cytokine receptors and exist in six isoforms (LEPR a-f), and all the isoforms are encoded by LEPR gene; out of these isoforms, the LEPR-b receptor is the 'longest form,' and in most of the cases, mutations in this isoform cause severe obesity. Also, mutations in the leptin gene (LEP) or its receptors gene can lead to obesity. Some biochemical pathways affect the bioactivity of leptin and/or its receptors. To date, eleven pathogenic mutations have been reported in the LEP which are p.L72S, p.N103K, p.R105W, p.H118L, p.S141C, p.W121X c.104_106delTCA, c.135del3bp, c.398delG, c.481_482delCT, and c.163C>T. Different mutations in the LEPR have also been reported as c.2396-1 G>T, c.1675 G>A, p.P316T, etc. In some studies, where leptin was deficient, leptin replacement therapy has shown positive impact by preventing weight gain and obesity.

Autosomal recessive Stickler syndrome due to a loss of function mutation in the COL9A3 gene.

Stickler syndrome (STL) is a clinically variable and genetically heterogeneous syndrome characterized by ophthalmic, articular, orofacial, and auditory manifestations. STL has been described with both autosomal dominant and recessive inheritance. The dominant form is caused by mutations of COL2A1 (STL 1, OMIM 108300), COL11A1 (STL 2, OMIM 604841), and COL11A2 (STL 3, OMIM 184840) genes, while recessive forms have been associated with mutations of COL9A1 (OMIM 120210) and COL9A2 (OMIM 120260) genes. Type IX collagen is a heterotrimeric molecule formed by three genetically distinct chains: α1, α2, and α3 encoded by the COL9A1, COL9A2, and COL9A3 genes. Up to this time, only heterozygous mutations of COL9A3 gene have been reported in human and related to: (1) multiple epiphyseal dysplasia type 3, (2) susceptibility to an intervertebral disc disease, and (3) hearing loss. Here, we describe the first autosomal recessive Stickler family due to loss of function mutations (c.1176_1198del, p.Gln393Cysfs*25) of COL9A3 gene. These findings extend further the role of collagen genes family in the disease pathogenesis.

Entire CAPN3 gene deletion in a patient with limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A) due to mutations in the CAPN3 gene is one of the most common of autosomal recessive limb-girdle muscular dystrophies. We describe a patient who had a typical LGMD2A phenotype and posterior compartment involvement on MRI. Different genetic analyses were performed, including microarray analysis. There was an apparently homozygous mutation in exon 24, c.2465G>T, p.(*822Leuext62*), and a lack of correlation in the disease segregation analyses. This suggested the presence of a genomic rearrangement. In fact, a heterozygous deletion of the entire CAPN3 gene was found. This novel deletion comprised the terminal region of the GANC gene and the entire CAPN3 gene. This finding points out the need to reconsider and adapt our current strategy of molecular diagnosis in order to detect these types of genomic rearrangements that escape standard mutation screening procedures.