Four novel GALC gene mutations in two Chinese patients with Krabbe disease

Krabbe disease (OMIM #245200) is a rare autosomal recessive leukodystrophy caused by deficiency of galactocerebrosidase (GALC) activity. We identified four novel mutations of the GALC gene in two unrelated Chinese families with Krabbe disease: one insertion mutation, c.1836_1837insT, and one nonsense mutation, c.599C>A (p.S200X), in an infantile patient, and one deletion mutation, c.1911+1_1911+5delGTAAG, and one missense mutation, c.2041G>A, in an adult late-onset patient. This is the first identification of GALC mutations in the Chinese population.     

Late-onset Krabbe disease is predominant in Japan and its mutant precursor protein undergoes more effective processing than the infantile-onset form

Krabbe disease is an autosomal recessive leukodystrophy caused by the deficiency of the galactocerebrosidase (GALC) enzyme. It is pathologically characterized by demyelination of the central and peripheral nervous systems by accumulation of galactosylsphingosine. To date, more than 120 mutations in the GALC gene have been reported worldwide and genotype–phenotype correlations have been reported in some types of mutations. In this study, we analyzed 22 unreported Japanese patients with Krabbe disease and summarized a total of 51 Japanese patients, including 29 previously reported patients. To elucidate how GALC mutations impair enzymatic activity, multiple disease-causing mutations including common mutations and polymorphisms were investigated for enzymatic activity and precursor processing ability with transient expression system. We also performed 3-D enzyme structure analysis to determine the effect of each new mutation. Five novel mutations were detected including one deletion c.1808delT [p.L603X], one nonsense mutation c.1023C>G [p.Y341X], and three missense mutations c.209T>C [p.L70P], c.1054G>A [p.G352R], and c.1937G>C [p.G646A]. For the total of 51 patients, 59% had late-onset forms of Krabbe disease. Seven common mutations accounted for 58% of mutant alleles of patients with Krabbe disease in Japan. Infantile-onset mutations had almost no enzyme activity, while late-onset mutations had 4%–20% of normal enzyme activity. The processing rate of precursor GALC protein to mature form was slower for infantile-onset mutations. Heat stability of the mutant proteins revealed that p.G270D was more stable compared to the other mutations. The constructed 3D-model showed that the residues for Krabbe mutations were less solvent-accessible and located in the core region of GALC protein. In conclusion, we have demonstrated that the most common phenotype in Japan is the late-onset type, that the enzyme activity for GALC mutants is correlated with mutational severity, and that the most pathogenic factor is due to the processing rate from the precursor to the mature protein.     

Molecular heterogeneity of late-onset forms of globoid-cell leukodystrophy.

Globoid-cell leukodystrophy (GLD) is an autosomal recessive inherited disorder caused by the deficiency of galactocerebrosidase, the lysosomal enzyme responsible for the degradation of the myelin glycolipid galactocerebroside. Although the most common form of the disease is the classical infantile form (Krabbe disease), later-onset forms also have been described. We have analyzed the galactocerebrosidase gene in 17 patients (nine families) with late-onset GLD and in 1 patient with classical Krabbe disease. Half of the patients were heterozygous for the large gene deletion associated with the 502C-->T polymorphism, the most common mutation in infantile patients. Several novel mutations that result in deficient galactocerebrosidase activity were also identified in these patients. They include the missense mutations R63H, G95S, M101L, G268S, Y298C, and I234T; the nonsense mutation S7X; a one-base deletion (805delG); a mutation that interferes with the splicing of intron 1; and a 34-nt insertion in the RNA, caused by the aberrant splicing of intron 6. All of these genetic defects are clustered in the first 10 exons of the galactocerebrosidase gene and therefore affect the 50-kD subunit of the mature enzyme. Studies on the distribution and enzymatic activity of the polymorphic alleles 1637T/C (I546/T546) provided support for previous data that had indicated the existence of two galactocerebrosidase forms with different catalytic activities in the general population. Our data also indicate that the mutations occur preferentially in the "low activity" 1637C allele.     

Two different mutations are responsible for Krabbe disease in the Druze and Moslem Arab populations in Israel

Infantile Krabbe disease is a severe, fatal autosomal recessive disorder resulting from the deficiency of galactocerebrosidase (GALC) activity. It is relatively common in two separate inbred communities in Israel. In the Druze community in Northern Israel and two Moslem Arab villages located near Jerusalem the incidence of Krabbe disease is about 1 in 100–150 live births. With our cloning of the GALC gene, mutation analysis of these populations was undertaken. The Moslem Arabs were homozygous for two mutations in the GALC gene; a T-to-C transition at cDNA position 1637 (counting from the A of the initiation codon), which is considered a polymorphism, and a G-to-A transition at position 1582, which changes the codon for aspartic acid to one for asparagine. The Druze patients are homozygous for a T-to-G transversion at position 1748, which changes the codon for isoleucine to one for serine. Expression studies confirmed the deleterious nature of these mutations. The development of a simple polymerase chain reaction (PCR) amplification and restriction enzyme digestion method to identify these alleles will lead to accurate carrier testing and improved genetic counseling for interested individuals in these communities.     

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

Mutation spectrum of phenylketonuria in Syrian population: Genotype–phenotype correlation

Characterization of the molecular basis of phenylketonuria (PKU) in Syria has been accomplished through the analysis of 78 unrelated chromosomes from 39 Syrian patients with PKU. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by using molecular detection methods based on the restriction fragment length polymorphism (RFLP), artificial constructed restriction sites (ACRS) PCR and direct DNA sequencing. 56.4% of the patients had cPKU. A mutation detection rate of 79.49% was achieved and sixteen different mutations were found: missense 56.25%, splice site 37.5%, and frameshift 6.25%. The predominant mutation in this population sample was p.R261Q G>A, p.F55>Lfs and p.R243Q G>A. No mutation in six PKU patients was observed. In 57.9% of patient genotypes, the metabolic phenotype could be predicted. The identification of the mutations in the PAH gene and the genotype–phenotype correlation should facilitate the evaluation of metabolic phenotypes, diagnosis, implementation of optimal dietary therapy, and determination of prognosis in the patients and genetic counseling for the patient's relatives.     

The novel heterozygous Thr377Arg MYOC mutation causes severe Juvenile Open Angle Glaucoma in a large Pakistani family

Glaucoma is one of the major causes of blindness worldwide with characteristic optic disc changes and elevated intraocular pressure. It is subcategorized into Primary Open Angle Glaucoma (POAG) and Juvenile Open Angle Glaucoma (JOAG) depending upon age of the disease onset. Myocilin (MYOC) is the frequently mutated gene in familial cases of glaucoma. MYOC mutations show variable phenotype and penetrance. This study was aimed to identify disease causing mutation in 8 affected of a consanguineous family diagnosed with severe form of Juvenile Open Angle Glaucoma. Homozygosity mapping with four microsatellite markers and subsequent direct sequencing of MYOC revealed a novel heterozygous transition c.1130 C>G, substituting Threonine in to Arginine at codon 377 (p.Thr377Arg) of MYOC. This mutation was segregating with phenotype in all affected and was not found in control subjects. Ophthalmological findings revealed JOAG with severe and rapidly progressive phenotype. The age of onset was in the first decade of life and maximum Intra Ocular Pressure (IOP) recorded was 25 mm Hg. Bioinformatic tools predicted C to G transition at c.1130 as pathogenic and no structural changes were predicted in protein. This is the first report of novel MYOC mutation from Pakistan; segregating as autosomal dominant trait in large family diagnosed with JOAG. Identification of novel disease causing allele in MYOC indicates genetic heterogeneity of the population. This finding will help to provide genetic counseling to the affected family and carriers of this mutation may be advised for early therapeutic intervention to avoid irreversible visual loss.     

Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases

Background

Inherited retinal disorders are clinically and genetically heterogeneous with more than 150 gene defects accounting for the diversity of disease phenotypes. So far, mutation detection was mainly performed by APEX technology and direct Sanger sequencing of known genes. However, these methods are time consuming, expensive and unable to provide a result if the patient carries a new gene mutation. In addition, multiplicity of phenotypes associated with the same gene defect may be overlooked.

Methods

To overcome these challenges, we designed an exon sequencing array to target 254 known and candidate genes using Agilent capture. Subsequently, 20 DNA samples from 17 different families, including four patients with known mutations were sequenced using Illumina Genome Analyzer IIx next-generation-sequencing (NGS) platform. Different filtering approaches were applied to identify the genetic defect. The most likely disease causing variants were analyzed by Sanger sequencing. Co-segregation and sequencing analysis of control samples validated the pathogenicity of the observed variants.

Results

The phenotype of the patients included retinitis pigmentosa, congenital stationary night blindness, Best disease, early-onset cone dystrophy and Stargardt disease. In three of four control samples with known genotypes NGS detected the expected mutations. Three known and five novel mutations were identified in NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 and CACNA1F. One of the control samples with a known genotype belongs to a family with two clinical phenotypes (Best and CSNB), where a novel mutation was identified for CSNB. In six families the disease associated mutations were not found, indicating that novel gene defects remain to be identified.

Conclusions

In summary, this unbiased and time-efficient NGS approach allowed mutation detection in 75% of control cases and in 57% of test cases. Furthermore, it has the possibility of associating known gene defects with novel phenotypes and mode of inheritance.     

A novel RAB7 mutation in a Chinese family with Charcot–Marie–Tooth type 2B disease

Charcot–Marie–Tooth type 2B (CMT2B) disease is a hereditary motor and sensory neuropathy subtype characterized by prominent loss of sensation, distal muscle weakness and wasting skin ulcers. Recurrent ulcers often require amputation of lower limbs. To date, only four mutations of the RAB7 gene, which encodes the small GTPase, have been associated with CMT2B. A Chinese family with CMT2B was identified. Direct DNA sequencing performed on the affected individuals in this family revealed a novel mutation (p.Asn161Ile) in RAB7. The mutation is located in a potential mutational hotspot region, implicating the importance of this region for RAB7 protein. This is the first report of RAB7 mutation in Asian population.     

A Novel PRPF31 Mutation in a Large Chinese Family with Autosomal Dominant Retinitis Pigmentosa and Macular Degeneration

Purpose

This study was intended to identify the disease causing genes in a large Chinese family with autosomal dominant retinitis pigmentosa and macular degeneration.

Methods

A genome scan analysis was conducted in this family for disease gene preliminary mapping. Snapshot analysis of selected SNPs for two-point LOD score analysis for candidate gene filter. Candidate gene PRPF31 whole exons'' sequencing was executed to identify mutations.

Results

A novel nonsense mutation caused by an insertion was found in PRPF31 gene. All the 19 RP patients in 1085 family are carrying this heterozygous nonsense mutation. The nonsense mutation is in PRPF31 gene exon9 at chr19:54629961-54629961, inserting nucleotide “A” that generates the coding protein frame shift from p.307 and early termination at p.322 in the snoRNA binding domain (NOP domain).

Conclusion

This report is the first to associate PRPF31 gene''s nonsense mutation and adRP and JMD. Our findings revealed that PRPF31 can lead to different clinical phenotypes in the same family, resulting either in adRP or syndrome of adRP and JMD. We believe our identification of the novel “A” insertion mutation in exon9 at chr19:54629961-54629961 in PRPF31 can provide further genetic evidence for clinical test for adRP and JMD.     

A Novel Missense SNRNP200 Mutation Associated with Autosomal Dominant Retinitis Pigmentosa in a Chinese Family

The SNRNP200 gene encodes hBrr2, a helicase essential for pre-mRNA splicing. Six mutations in SNRNP200 have recently been discovered to be associated with autosomal dominant retinitis pigmentosa (adRP). In this work, we analyzed a Chinese family with adRP and identified a novel missense mutation in SNRNP200. To identify the genetic defect in this family, exome of the proband was captured and sequencing analysis was performed to exclude known genetic defects and find possible pathogenic mutations. Subsequently, candidate mutations were validated in affected family members using Sanger sequencing. A novel missense mutation, c.2653C>G transition (p.Q885E), in exon 20 of SNRNP200 was identified. The mutation co-segregated with the disease phenotype over four generations and was absent in 100 normal unaffected individuals. This mutation occurs at highly conserved position in hBrr2 and is predicted to have a functional impact, suggesting that hBrr2-dependent small nuclear riboproteins (snRNPs) unwinding and spliceosome activation is important in the pathogenesis of some variants of RP.     

Clinical and molecular genetic study of infantile-onset Pompe disease in Chinese patients: Identification of 6 novel mutations

Pompe disease is an autosomal recessive disorder and is caused by a deficiency in acid alpha-glucosidase (GAA). A broad range of studies have been performed on Pompe patients from different countries. However, the clinical course and molecular basis of the disease in Mainland China have not been well defined. In the present study, we examined a total of 18 Chinese children with infantile-onset Pompe disease to better understand the clinical and genetic features in this population. The median age at symptom onset was 3.6 months (range: 1.7–6.8 months) and 6.3 months at diagnosis (range: 2.5–9.3 months). All but 1 patient died at a median age of 8.2 months (range: 4.7–18.7 months). Molecular analysis revealed 20 different mutations, 6 of which are novel (c.1356delC, c.378G > A, c.1827C > G, c.859-2 A > T, c.1551 + 2T > G, and c.1465G > T). The most common mutation in the study was c.1935C > A, accounting for 25% (9/36 alleles) of the mutations. Our study provides the first comprehensive examination of the clinical course of infantile-onset Pompe disease and mutations of the GAA gene for patients in Mainland China. Our results confirm the high prevalence of the c.1935C > A mutation, previously reported for other populations, in Mainland Chinese patients with infantile-onset Pompe disease. Furthermore, six novel mutations in the GAA gene are reported for the first time.     

Targeted next-generation sequencing identifies ABCA4 mutations in Chinese families with childhood-onset and adult-onset Stargardt disease

Background: Stargardt disease (STGD) is the most common form of juvenile macular dystrophy associated with progressive central vision loss, and is agenetically and clinically heterogeneous disease. Molecular diagnosis is of great significance in aiding the clinical diagnosis, helping to determine the phenotypic severity and visual prognosis. In the present study, we determined the clinical and genetic features of seven childhood-onset and three adult-onset Chinese STGD families. We performed capture next-generation sequencing (NGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes.Methods: In all, ten unrelated Chinese families were enrolled. Panel-based NGS was performed to identify potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes, including the five known STGD genes (ABCA4, PROM1, PRPH2, VMD2, and ELOVL4). Variant analysis, Sanger validation, and segregation tests were utilized to validate the disease-causing mutations in these families.Results: Using systematic data analysis with an established bioinformatics pipeline and segregation analysis, 17 pathogenic mutations in ABCA4 were identified in the 10 STGD families. Four of these mutations were novel: c.371delG, c.681T > G, c.5509C > T, and EX37del. Childhood-onset STGD was associated with severe visual loss, generalized retinal dysfunction and was due to more severe variants in ABCA4 than those found in adult-onset disease.Conclusions: We expand the existing spectrum of STGD and reveal the genotype–phenotype relationships of the ABCA4 mutations in Chinese patients. Childhood-onset STGD lies at the severe end of the spectrum of ABCA4-associated retinal phenotypes.     

The WNT7A G204S mutation is associated with both Al-Awadi–Raas Rothschild syndrome and Fuhrmann syndrome phenotypes

Two syndromes are known to be associated with WNT7A mutations: Al-Awadi–Raas-Rothschild syndrome (AARRS) and Fuhrmann syndrome. Woods et al. (2006) showed that there is complete and partial loss of WNT7A function in these two syndromes respectively. Therefore, both syndromes have similar clinical features but the phenotype in Fuhrmann syndrome is less severe. The G204S mutation was previously reported to result in AARRS phenotype in three Saudi families. In the current communication, we report on a different unrelated Saudi patient with the same mutation but the patient had Fuhrmann syndrome phenotype. We believe this case is important because it questions the presence of a phenotype–genotype correlation in WNT7A mutations and because it demonstrates that the G204S mutation may be associated with both AARRS and Fuhrmann phenotypes.     

Identification of one novel mutation in the EVC2 gene in a Chinese family with Ellis–van Creveld syndrome

Ellis–van Creveld syndrome (EvC) is a rare autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly, and dysplastic nails and teeth. It is caused by biallelic mutations in the EVC or EVC2 gene. Here, we identified a novel nonsense mutation p.W828X (c.2484G>A) in exon 14 and a recurrent nonsense mutation p. R399X (c.1195C>T) in exon 10 of EVC2 gene in a Chinese boy with EvC. Identification of a novel genotype in EvC will provide clues to the phenotype–genotype relations and may assist not only in the clinical diagnosis of EvC but also in the interpretation of genetic information used for prenatal diagnosis and genetic counseling.     

Two novel isovaleryl-CoA dehydrogenase gene mutations in a Chinese infant

Isovaleric acidemia (IVA) is a rare inherited metabolic disease caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD). Newborn screening with tandem mass spectrometry leads to early identification of individuals with risk of IVA. The family specific mutations are useful for prenatal diagnosis. Molecular genetic analysis helps to further confirm the clinical diagnosis of IVA. We describe here the clinical and metabolic features of a Chinese infant with early onset IVA. Sequence analysis of the IVD gene identifies compound heterozygous mutations in this patient, c.39G > A (p.W13X) nonsense mutation and c.597C > G (p.I199M) missense mutation, both of which are previously unreported. Structural analyses suggest that the p.I199M missense mutation may destabilize the IVD monomer structure and affect the interaction between IVD and flavin adenine dinucleotide. Both the clinical and genetic features of this patient help to further expand our knowledge of IVA.     

Identification of Two Novel Mutations in the PHEX Gene in Chinese Patients with Hypophosphatemic Rickets/Osteomalacia

Objective

X-linked dominant hypophosphatemia (XLH) is the most prevalent form of inherited rickets/osteomalacia in humans. The aim of this study was to identify PHEX gene mutations and describe the clinical features observed in 6 unrelated Chinese families and 3 sporadic patients with hypophosphatemic rickets/osteomalacia.

Methods

For this study, 45 individuals from 9 unrelated families of Chinese Han ethnicity (including 16 patients and 29 normal phenotype subjects), and 250 healthy donors were recruited. All 22 exons and exon-intron boundaries of the PHEX gene were amplified by polymerase chain reaction (PCR) and directly sequenced.

Results

The PHEX mutations were detected in 6 familial and 3 sporadic hypophosphatemic rickets/osteomalacia. Altogether, 2 novel mutations were detected: 1 missense mutation c.1183G>C in exon 11, resulting in p.Gly395Arg and 1 missense mutation c.1751A>C in exon 17, resulting in p.His584Pro. No mutations were found in the 250 healthy controls.

Conclusions

Our study increases knowledge of the PHEX gene mutation types and clinical phenotypes found in Chinese patients with XLH, which is important for understanding the genetic basis of XLH. The molecular diagnosis of a PHEX genetic mutation is of great importance for confirming the clinical diagnosis of XLH, conducting genetic counseling, and facilitating prenatal intervention, especially in the case of sporadic patients.     

A Novel WRN Frameshift Mutation Identified by Multiplex Genetic Testing in a Family with Multiple Cases of Cancer

Next-generation sequencing technology allows simultaneous analysis of multiple susceptibility genes for clinical cancer genetics. In this study, multiplex genetic testing was conducted in a Chinese family with multiple cases of cancer to determine the variations in cancer predisposition genes. The family comprises a mother and her five daughters, of whom the mother and the eldest daughter have cancer and the secondary daughter died of cancer. We conducted multiplex genetic testing of 90 cancer susceptibility genes using the peripheral blood DNA of the mother and all five daughters. WRN frameshift mutation is considered a potential pathogenic variation according to the guidelines of the American College of Medical Genetics. A novel WRN frameshift mutation (p.N1370Tfs*23) was identified in the three cancer patients and in the youngest unaffected daughter. Other rare non-synonymous germline mutations were also detected in DICER and ELAC2. Functional mutations in WRN cause Werner syndrome, a human autosomal recessive disease characterized by premature aging and associated with genetic instability and increased cancer risk. Our results suggest that the WRN frameshift mutation is important in the surveillance of other members of this family, especially the youngest daughter, but the pathogenicity of the novel WRN frameshift mutation needs to be investigated further. Given its extensive use in clinical genetic screening, multiplex genetic testing is a promising tool in clinical cancer surveillance.