Mutation In Compound Heterozygosity Research Articles

Generation of two induced pluripotent stem cell lines (CHOCi002-A and CHOCi003-A) from Pompe disease patients with compound heterozygous mutations in the GAA gene

Pompe disease is an autosomal recessive lysosomal storage disease caused by pathogenic variants in GAA, which encodes an enzyme integral to glycogen catabolism, acid α-glucosidase. Disease-relevant cell lines are necessary to evaluate the efficacy of genotype-specific therapies. Dermal fibroblasts from two patients presenting clinically with Pompe disease were reprogrammed to induced pluripotent stem cells using the Sendai viral method. One patient is compound heterozygous for the c.258dupC (p.N87QfsX9) frameshift mutation and the c.2227C>T (p.Q743X) nonsense mutation. The other patient harbors the c.-32–13T>G splice variant and the c.1826dupA (p.Y609X) frameshift mutation in compound heterozygosity.

P.226 - Late-onset limb-girdle myopathy with oculobulbar signs and rimmed vacuoles associated with a novel Pompe disease mutation

Pompe disease (PD) is an autosomal recessive lysosomal storage disorder caused by acid alfa-glucosidase (GAA) deficiency. Late-onset phenotype is heterogeneous, and it may include ptosis and/or bulbar weakness. Since 2006 enzyme replacement therapy with alglucosidase alfa has become available with the best benefit depending on residual muscle function. Therefore, its prompt diagnosis has become more noteworthy. We describe a 50-year-old Portuguese woman, which presented with slowly progressive speech slurring and limb-girdle muscle weakness for the previous 25 years. In the last 10 years she also complained of progressive left eyelid drop. No diplopia or dysphagia was present. There was no fatigability or diurnal fluctuation in symptoms. Her medical and familial history was unremarkable. Neurological examination disclosed left ptosis, moderate tongue atrophy and weakness, slight dysarthria and severe limb-girdle muscle weakness. A mildly restrictive respiratory pattern was disclosed on pulmonary function tests. There was no heart involvement. Nerve conduction studies were normal. Needle electromyography disclosed myopathic potentials associated with fibrillation potentials and positive sharp waves. Repetitive nerve stimulation was unremarkable. Serum creatine kinase was mildly elevated (648–881 UI/L). Muscle biopsy showed myopathic changes with some rimmed vacuoles. There was no excessive glycogen accumulation seen with PAS and by electron microscopy (EM). There were no filament inclusions under EM. A reduced GAA enzyme activity (1.58 pmol/h/punction; normal >4.51) was disclosed on dried blood spot analysis. Sequence analysis of the GAA gene revealed a novel pathological mutation in compound heterozygosity [c.-32-13T > G/c.2319C > G(p.Y773*)]. Six months after enzyme replacement therapy, ptosis improved and respiratory function remained stable. This case highlights the need for a high index of suspicion for PD in adult patients with unexplained oculobulbar and limb-girdle muscle weakness, even if the muscle biopsy features are completely atypical.

Compound heterozygous MYO7A mutations segregating Usher syndrome type 2 in a Han family

ObjectiveIdentification of rare deafness genes for inherited congenital sensorineural hearing impairment remains difficult, because a large variety of genes are implicated. In this study we applied targeted capture and next-generation sequencing to uncover the underlying gene in a three-generation Han family segregating recessive inherited hearing loss and retinitis pigmentosa. MethodsAfter excluding mutations in common deafness genes GJB2, SLC26A4 and the mitochondrial gene, genomic DNA of the proband of a Han family was subjected to targeted next-generation sequencing. The candidate mutations were confirmed by Sanger sequencing and subsequently analyzed with in silico tools. ResultsAn unreported splice site mutation c.3924+1G > C compound with c.6028G > A in the MYO7A gene were detected to cosegregate with the phenotype in this pedigree. Both mutations, located in the evolutionarily conserved FERM domain in myosin VIIA, were predicted to be pathogenic. In this family, profound sensorineural hearing impairment and retinitis pigmentosa without vestibular disorder, constituted the typical Usher syndrome type 2. ConclusionIdentification of novel mutation in compound heterozygosity in MYO7A gene revealed the genetic origin of Usher syndrome type 2 in this Han family.

Isolated p.H62L Mutation in theCYP21A2Gene in a Simple Virilizing 21-Hydroxylase Deficient Patient

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90%–95% of cases. This autosomal recessive disorder has a broad spectrum of clinical forms, ranging from severe or classical, which includes the salt-wasting and simple virilizing forms, to the mild late onset or nonclassical form. Most of the disease-causing mutations described are likely to be the consequence of nonhomologous recombination or gene conversion events between the active CYP21A2 gene and its homologous CYP21A1P pseudogene. Nevertheless, an increasing number of naturally occurring mutations have been found. The change p.H62L is one of the most frequent rare mutations of the CYP21A2 gene. It was suggested that the p.H62L represents a mild mutation that may be responsible for a more severe enzymatic impairment when presented with another mild mutation on the same allele. In this report, a 20-year-old woman carrying an isolated p.H62L mutation in compound heterozygosity with c.283-13A/C>G mutation is described. Although a mildly nonclassical phenotype was expected, clinical signs and hormonal profile of the patient are consistent with a more severe simple virilizing form of 21-hydroxylase deficiency. The study of genotype-phenotype correlation in additional patients would help in defining the role of p.H62L in disease manifestation.

A novel p.Leu213X mutation in GJB2 gene in a Portuguese family

IntroductionHearing loss is the most common sensory disability and is present in about 1.9 per 1000 infants at birth. The DFNB1 locus (13q11-q12) includes the genes GJB2, coding for connexin 26, and GJB6, encoding connexin 30. More than 100 mutations have been identified associated with autosomal dominant and recessive hearing loss in the GJB2 gene. ObjectivesThe aim of the present study was to identify the genetic aetiology of deafness in two Portuguese individuals, presenting nonsyndromic sensorineural moderate and severe hearing loss, respectively. Patients and methodsThe individuals were evaluated in both ears by pure tone audiometry and blood samples were collected after written informed consent was signed. DNA extraction and PCR amplification of GJB2 coding region followed standard methodologies. PCR products were automatically sequenced in both directions. ResultsWe identified a novel mutation, c.638T>A (p.Leu213X), in GJB2 gene. This nonsense mutation was found in both siblings, and was inherited from their hearing father. Molecular analysis showed that the two siblings were also heterozygous for c.333-334delAA, a previously described GJB2 deletion. This novel mutation was not found in a random control sample of 480 individuals that were screened for coding region of GJB2 gene. p.Leu213X mutation identified in this study for the first time changes the codon 213, coding for a highly conserved and slowly evolving residue of connexin 26, localised to the C-terminus domain of the protein, to a STOP codon, leading to the deletion of the last 14 amino acids of the protein. ConclusionWe can conclude that the aetiology of deafness in these individuals is due to the GJB2 genotype involving the c.333-334delAA deletion and the novel p.Leu213X mutation in compound heterozygosity.

Compound heterozygosity of the novel c.292C > T (p.R98W) and the c.35delG GJB2 mutations in postlingual, non-syndromic, sensorineural deafness

ObjectivesConnexins (Cxs) are membrane-spanning proteins that co-assemble into intercellular gap junction channels. Gap junction channels mediate electrical and biochemical communication between adjacent cells and play vital roles as mediators of intercellular molecular signaling. Cx-linked deafness highlights the key role of gap junctions in the physiological processes of hearing. Co-localization of Cxs with the gap junction system in the inner ear suggests a role in cochlear electrolyte homeostasis. During auditory transduction, they are proposed to maintain membrane potentials by regulating the flow of potassium ions between the sensory epithelia of the inner ear. MethodsClinical and molecular genetic methods were employed in a Greek proband presenting with bilateral, postlingual, non-syndromic, sensorineural deafness. ResultsWe detected a novel c.292C>T (p.R98W) mutation in compound heterozygosity with the c.35delG mutation in the GJB2 gene. ConclusionAlthough mutations in the GJB2 gene usually cause prelingual, severe to profound deafness, compound heterozygosity of the novel c.292C>T (p.R98W) and the c.35delG GJB2 mutations appears to be the cause of postlingual, moderate, sensorineural deafness in our proband.

Clinical characteristics and molecular analysis of three Chinese children with Omenn syndrome

Omenn syndrome (OS) is a rare autosomal recessive genetic disorder and presents symptoms of severe combined immunodeficiency characterized by erythrodermia, eosinophilia, hepatosplenomegaly, lymphadenopathy, and elevated serum IgE levels. OS has been found to be caused by mutations in RAG1 or RAG2 gene that result in partial V(D)J recombination activity. No study on OS has been reported in Chinese children so far. In this study, the genotype and phenotypes of three infants with OS from three unrelated Chinese families were investigated. All the three children had most of the characteristics of OS except normal serum IgE level. Compound heterozygosity mutations in RAG1 gene (1983 G>A; 2444 C>T and 2219 C>T; 3127 C>G) were identified in two cases, and a homozygous deletion mutation with a premature stop codon was found at residue 2302 of RAG1 gene (2302delT, I729X) in the remaining case, including three novel mutations (2302delT, I729X; 2219 C>T, R699W; and 3127 C>G, Y1001X). Spectratyping analysis of T-cell receptor β-chain variable region (TCRVβ) gene rearrangement was performed in case 1 and case 2. All the 25 TCRVβ subfamilies presented monoclonal or oligoclonal peaks in case 1 and 11 TCRVβ subfamilies were very weak or even absent in case 2. This was the first report about OS in Chinese children. Molecular genetic testing represents an important tool for early confirmed diagnosis and may allow accurate carrier detection and prenatal diagnosis.

Triose phosphate isomerase deficiency associated with two novel mutations in TPI gene

We report the clinical, haematological and molecular characteristics of two triose phosphate isomerase deficient patients affected by haemolytic anaemia and neuromuscular impairment. The sequence of complete TPI gene showed the presence of two previously undescribed mutations: c.722 T>C (Phe240Ser) and c.28 insG; each of the two unrelated patients showed the new mutation in compound heterozygosity with the most common variant Glu104Asp. The association of Glu104Asp with c.28 insG resulted in a very severe clinical pattern.

Unique mixed phenotype and unexpected functional effect revealed by novel compound heterozygosity mutations involving SCN5A

Functional characterization of mutations involving the SCN5A-encoded cardiac sodium channel has established the pathogenic mechanisms for type 3 long QT syndrome and type 1 Brugada syndrome and has provided key insights into the physiological importance of essential structure-function domains. This study sought to present the clinical and biophysical phenotypes discerned from compound heterozygosity mutations in SCN5A on different alleles in a toddler diagnosed with QT prolongation and fever-induced ventricular arrhythmias. A 22-month-old boy presented emergently with fever and refractory ventricular tachycardia. Despite restoration of sinus rhythm, the infant sustained profound neurological injury and died. Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, comprehensive open-reading frame/splice mutational analysis of the 12 known long QT syndrome susceptibility genes was performed. The infant had 2 SCN5A mutations: a maternally inherited N-terminal frame shift/deletion (R34fs/60) and a paternally inherited missense mutation, R1195H. The mutations were engineered by site-directed mutagenesis and heterologously expressed transiently in HEK293 cells. As expected, the frame-shifted and prematurely truncated peptide, SCN5A-R34fs/60, showed no current. SCN5A-R1195H had normal peak and late current but abnormal voltage-dependent gating parameters. Surprisingly, co-expression of SCN5A-R34fs/60 with SCN5A-R1195H elicited a significant increase in late sodium current, whereas co-expression of SCN5A-WT with SCN5A-R34fs/60 did not. A severe clinical phenotype characterized by fever-induced monomorphic ventricular tachycardia and QT interval prolongation emerged in a toddler with compound heterozygosity involving SCN5A: R34fs/60, and R1195H. Unexpectedly, the 94-amino-acid fusion peptide derived from the R34fs/60 mutation accentuated the late sodium current of R1195H-containing Na(V)1.5 channels in vitro.

Microconversion betweenCYP21A2andCYP21A1PPromoter Regions Causes the Nonclassical Form of 21-Hydroxylase Deficiency

Most mutations causing 21-hydroxylase deficiency originate from microconversions between CYP21 pseudogenes and active genes. However, around 20% of the alleles in the nonclassical form (NC-21OHD) remain without identified mutations, suggesting the involvement of regulatory regions. The pseudogene promoter is 80% less active than the CYP21A2 due to the presence of -126C>T, -113G>A, -110T>C, and -103A>G mutations. Additionally, mutations in the steroidogenic factor-1 binding sites of the CYP21 distal regulatory region, located at 4676 bases upstream from the cap site of the CYP21A2 gene, decrease its transcription to 35%. The objective of the study was to investigate the CYP21A2 promoter/regulatory regions in NC-21OHD patients with undetermined genotype. The study included 17 NC-21OHD patients and 50 controls. Promoter/regulatory regions were sequenced from peripheral leukocytes' genomic DNA. The identified substitutions were evaluated through EMSA using -132/-97 wild-type and mutant probes and nuclear extracts from NCI-H295A cells. Transcriptional activity studies were performed with wild-type and mutant constructions transfected in NCI-H295A cells. No mutations were identified in the distal regulatory regions. The -126C>T, -113G>A, -110T>C promoter mutations were found in compound heterozygosity with the V281L mutation in one patient and the -126C>T mutation in compound heterozygosity with the I2 splice in another. The -126T mutation decreases the transcriptional activity to 52%, compatible with the patient's nonclassical phenotype. EMSA demonstrated that the -132/-121 region is important for the DNA interaction with the specificity protein-1 transcription factor. Microconversions between CYP21A2 and CYP21A1P promoters could be involved in the nonclassical phenotype. Therefore CYP21A2 promoter analysis should be included in genetic studies of 21OHD.

16. Late-onset GM2 gangliosidosis and other hexosaminidase mutations among Jews

Publisher Summary This chapter discusses the late-onset G M2 gangliosidosis and other Hexosaminidase mutation among Jews. Tay-Sachs Disease (TSD) is caused by absence of β-hexosaminidase (Hex) A, following which heat inactivation method (HIM) for quantifying Hex A using the synthetic substrate 4-MUG. The interaction between Hex A and G M2 ganglioside substrate is accomplished by a nonenzymatic protein, the G M2 activator. Hex A-deficient adults have residual Hex A activity that are significantly higher in TSD patients, but significantly lower than in TSD heterozygotes. AGG is a slowly progressing neurodegenerative disorder. The patients have very low Hex A activity and usually carry the Gly269Ser mutation in compound heterozygosity with an infantile TSD mutation. Homozygotes for the Gly269Ser mutation are only mildly affected. The enzyme β-hexosaminidase (Hex) B does not degrade the G M2 ganglioside in humans, and therefore has no direct involvement in TSD. However, it is a major participant in the routine enzymatic assay for identification of TSD genotypes because Hex B, like Hex A, hydrolyzes the synthetic substrate 4-MUG, and, unlike Hex A, it is heat stabile. It is estimated that 1 in 140 Moroccans is a TSD carrier, and seven different TSD mutations in this community have been identified.

P.3.052 - First episode schizophrenia: Treatment and outcome

This chapter discusses the late-onset GM2 gangliosidosis and other Hexosaminidase mutation among Jews. Tay-Sachs Disease (TSD) is caused by absence of β-hexosaminidase (Hex) A, following which heat inactivation method (HIM) for quantifying Hex A using the synthetic substrate 4-MUG. The interaction between Hex A and GM2 ganglioside substrate is accomplished by a nonenzymatic protein, the GM2 activator. Hex A-deficient adults have residual Hex A activity that are significantly higher in TSD patients, but significantly lower than in TSD heterozygotes. AGG is a slowly progressing neurodegenerative disorder. The patients have very low Hex A activity and usually carry the Gly269Ser mutation in compound heterozygosity with an infantile TSD mutation. Homozygotes for the Gly269Ser mutation are only mildly affected. The enzyme β-hexosaminidase (Hex) B does not degrade the GM2 ganglioside in humans, and therefore has no direct involvement in TSD. However, it is a major participant in the routine enzymatic assay for identification of TSD genotypes because Hex B, like Hex A, hydrolyzes the synthetic substrate 4-MUG, and, unlike Hex A, it is heat stabile. It is estimated that 1 in 140 Moroccans is a TSD carrier, and seven different TSD mutations in this community have been identified.