Molecular defects identified by whole exome sequencing in a child with Fanconi anemia

Fanconi anemia is a rare genetic disease characterized by bone marrow failure, multiple congenital malformations, and an increased susceptibility to malignancy. At least 15 genes have been identified that are involved in the pathogenesis of Fanconi anemia. However, it is still a challenge to assign the complementation group and to characterize the molecular defects in patients with Fanconi anemia. In the current study, whole exome sequencing was used to identify the affected gene(s) in a boy with Fanconi anemia. A recurring, non-synonymous mutation was found (c.3971C>T, p.P1324L) as well as a novel frameshift mutation (c.989_995del, p.H330LfsX2) in FANCA gene. Our results indicate that whole exome sequencing may be useful in clinical settings for rapid identification of disease-causing mutations in rare genetic disorders such as Fanconi anemia.     

Mutation c.359_363delGTATTinsATAC in the COL4A5 Causes alport syndrome in a Chinese family

The X-linked form of Alport syndrome is associated with mutations in the COL4A5 gene, which is located at Xq22.3 and encodes the α5 chain of type IV collagen. Here we clinically characterized a Chinese family with Alport Syndrome, but no ocular or hearing abnormalities have been observed in any patient in the family. Through Linkage analysis and direct DNA sequencing, a novel complex deletion/insertion mutation c.359_363delGTATTinsATAC in the COL4A5 gene was identified in the family. The mutation was found in all affected family members, but was not present in the unaffected family individuals or the 200 controls. The predicted mutant protein in the family is a truncated protein consisting of only 153 residues. Our report for the first time revealed that the frameshift mutation in the type IV collagen chain α5 causes only renal disease, without extrarenal lesion. Our study broadens genotypic and phenotypic spectrum of COL4A5 mutations associated with Alport syndrome.     

WFS1 and non-syndromic low-frequency sensorineural hearing loss: A novel mutation in a Portuguese case

Low-frequency sensorineural hearing loss (LFSNHL) is an unusual type of HL in which frequencies at 2000 Hz and below are predominantly affected. Most of the families with LFSNHL carry missense mutations in WFS1 gene, coding for wolframin.     

Mutation spectrum of primary hyperoxaluria type 1 in Tunisia: Implication for diagnosis in North Africa

Background

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive inherited metabolic disease, characterized by progressive kidney failure due to renal deposition of calcium oxalate. Mutations in the AGXT gene, encoding the liver-specific enzyme alanine glyoxylate aminotransferase, are responsible for the disease. We aimed to determine the mutational spectrum causing PH1 and to provide an accurate tool for diagnosis as well as for prenatal diagnosis in the affected families.

Methods

Direct sequencing was used to detect mutations in the AGXT gene in DNA samples from 13 patients belonging to 12 Tunisian families.

Results

Molecular analysis revealed five mutations causing PH1 in Tunisia. The mutations were identified along exons 1, 2, 4, 5 and 7. The most predominant mutations were the Maghrebian “p.I244T” and the Arabic “p.G190R”. Furthermore, three other mutations characteristic of different ethnic groups were found in our study population. These results confirm the mutational heterogeneity related to PH1 in Tunisian population. All the mutations are in a homozygous state, reflecting the high impact of endogamy in our population.

Conclusion

Mutation analysis through DNA sequencing can provide a useful first line investigation for PH1. This identification could provide an accurate tool for prenatal diagnosis, genetic counseling and screen for potential presymptomatic individuals.     

A novel EFNB1 mutation in a patient with craniofrontonasal syndrome and right hallux duplication

Craniofrontonasal syndrome (CFNS, MIM #304110) is a rare X-linked dominant developmental disorder that shows paradoxically greater severity in affected females than in affected males. Our female patient with frontonasal dysplasia, craniosynostosis and additional malformations was consistent with CFNS. EFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases, is the only gene in which mutation is known to cause CFNS. Here, we describe 402 T > C, a novel de novo mutation on EFNB1. This mutation results in substitution of highly conserved isoleucine at 134th residue to threonine.     

New perspectives for pharmacological chaperoning treatment in methylmalonic aciduria cblB type

Methylmalonic aciduria cblB type (MMA cblB) is caused by the impairment of ATP:cob(I)alamin adenosyltransferase (ATR), the enzyme responsible for the synthesis of adenosylcobalamin (AdoCbl) from cob(I)alamin. No definitive treatment is available for patients with this condition and novel therapeutic strategies are therefore much needed. Recently, we described a proof-of-concept regarding the use of pharmacological chaperones as a treatment. This work describes the effect of two potential pharmacological chaperones - compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) and compound VI (4-(4-(4-fluorophenyl)-5-methyl-1H-pyrazol-3-yl)benzene-1,3-diol) - on six ATR mutants, including the most common, p.Arg186Trp. Comprehensive functional analysis identified destabilizing (p.Arg186Gln, p.Arg190Cys, p.Arg190His, p.Arg191Gln and p.Glu193Lys) and oligomerization (p.Arg186Trp and p.Arg191Gln) mutations. In a cellular model overexpressing the destabilizing/oligomerization mutations, compounds V and VI had a positive effect on the stability and activity of all ATR variants. When provided in combination with hydroxocobalamin a more positive effect was obtained than with the compounds alone, even in mutations previously described as B₁₂ non-responsive. In addition, a normal oligomerization profile was recovered after treatment of the p.Arg186Trp mutant with both compounds. These promising results confirm MMA cblB type as a conformational disorder and hence, pharmacological chaperones as a new therapeutic option alone or in combination with hydroxocobalamin for many patients with MMA cblB.     

UGT1A1 sequence variants associated with risk of adult hyperbilirubinemia: A quantitative analysis

Backgrounds and Aims

UDP-glucuronosyltransferase 1 A1 (UGT1A1) is an enzyme that transforms small lipophilic molecules into water-soluble and excretable metabolites. UGT1A1 polymorphisms contribute to hyperbilirubinemia. This study quantitatively associated UGT1A1 variants in patients with hyperbilirubinemia and healthy subjects.

Methods

A total of 104 individuals with hyperbilirubinemia and 105 healthy controls were enrolled for genotyping and DNA sequencing UGT1A1 sequence variants, including the Phenobarbital Response enhancer module (PBREM) region, the promoter region (TATA box), and the 5 exons for quantitative association with hyperbilirubinemia.

Results

Eleven UGT1A1 variants were revealed in the case and control subjects, four of which were novel coding variants. A variant of PBREM (UGT1A1*60) was found in 47.6% of the patients, a TA repeat motif in the 5-primer promoter region [A(TA)₇TAA,UGT1A1*28] was found in 27.9% of the patients, and p.G71R (UGT1A1*6) was in 33.2% of the patients. For the healthy controls, the frequency of UGT1A1*60, UGT1A1*28 and UGT1A1*6 was 26.7%, 9.0% and 15.7%, respectively. Homozygous UGT1A1*28 and homozygous UGT1A1*6 were significantly associated with the risk of adult hyperbilirubinemia, with an odds ratio (OR) of 17.79 (95% CIs, 2.11–133.61) and 14.93 (95% CIs, 1.83–121.88), respectively. Quantitative analysis showed that sense mutation (including UGT1A1*6) and UGT1A1*28/*28, but not UGT1A1*60/*60 or UGT1A1*1/*28, was associated with increased serum total bilirubin (TB) levels. High linkage disequilibrium occurred between UGT1A1*60 and UGT1A1*28 (D′ = 0.964, r² = 0.345).

Conclusions

This study identified four novel UGT1A1 coding variants, some of which were associated with increased serum TB levels. A quantitative approach to evaluate adult hyperbilirubinemia provides a more vigorous framework for better understanding of adult hyperbilirubinemia genetics.     

Mutation mapping of apolipoprotein A-I structure assisted with the putative cholesterol recognition regions

Twenty-nine from 52 missense mutations in apoA-I gene are predicted to be deleterious by both SIFT and PolyPhen-2 algorithms. Among those, eight mutations with a prominent change in structure stability as modeled by the SDM tool for both lipid-free (Mei and Atkinson (2011) PDB ID: 3R2P) and HDL-bound (Wu et al. (2009) PDB ID: 3K2S) apoA-I, are referred as structural. The remaining mutations with a preferential location in a long intrinsically disordered region, predicted by the SPINE-D and DNdisorder tools, may influence the functional sites. Among structural mutations, five amyloidosis-only-related mutations, significant in a lipid-free structure, are located in 1–90 region. Six amyloidosis- and hypoalphalipoproteinemia-associated mutations, differently significant in two chains of lipid-bound apoA-I, are distributed among the N-domain. Six cholesterol recognition putative motifs (5 CRAC/1 CCM) in apoA-I structure are suggested to interact with cholesterol. Among those, the K40-W50 partially conserved CCM sequence with a putative recognition feature, predicted by the MoRF tool, may underlie cholesterol binding to lipid-free apoA-I, the binding triggering the disorder-to-order transition within MoRF. Thus, the impairment of helix formation and accelerated protein aggregation may underlie the amyloidogenic effect of W50R substitution. Also, D102H substitution in conserved CRAC2 V97-K106 sequence may be harmful in reverse cholesterol transport. With PDBe Motifs and Sites algorithm, cholesterol is a ligand for L101, F104 and W108 residues in HDL-bound apoA-I. The influence of specific mutation on apoA-I structure and mutated apolipoprotein switch between different pathologies is suggested to depend on the surrounding phase properties.     

Complement component C6 deficiency in a Spanish family: Implications for clinical and molecular diagnosis

Complement component C6 deficiency is a genetic disease presenting as increased susceptibility to invasive Neisseria meningitidis infections. This disorder has rarely been diagnosed in the Spanish population. In this work we report the immunochemical and molecular characterization of complement C6 deficiency in a Spanish patient showing no detectable functional activity of either the classical or alternative complement pathways and reporting a history of several episodes of meningococcal meningitis. The levels of individual complement components C3, C4, C5, C7, C8 and C9 were within the normal range. However, C6 level was low in the patient's serum as measured by radial immunodiffusion. Exon-specific polymerase chain reaction and sequencing of the C6 gene revealed a previously described homozygous single base deletion in exon 6 (c.821delA), leading to a shift in the reading frame that caused the generation of a downstream stop codon, which, in turn, provoked the truncation of the C6 protein (p.Gln274fs). To our knowledge, this is the first report on the c.821delA mutation in the Spanish population, which has previously only been identified in individuals of African ancestry. Characterization of this mutation was thought interesting in order to elucidate its source and help understand the molecular basis of this uncommon deficiency in our population. Moreover, this report highlights the importance of complement screening in cases of repeated meningococcal infections in order to establish its involvement and to consider adequate clinical recommendations such as prophylactic antibiotics or meningococcal vaccines and, subsequently, for genetic counselling.