A novel homozygous KY variant causing a complex neurological disorder.

Mutations in the gene kyphoscoliosis peptidase (KY) are known to cause myofibrillar myopathy-7 and hereditary spastic paraplegia. We investigated the genetic cause of a complex neurological phenotype in a consanguineous Pakistani family with four affected members, manifesting lower limb spasticity and weakness, toe walking, pes equinovarus, and a speech disorder. Genome-wide linkage analysis with microsatellite markers delineated chromosome 3q22.2-q24 harboring the disease gene. Whole exome sequencing was performed for two subjects, identifying a homozygous 14-bp frameshift deletion NM_178554.6:c.842_855del; p(Val281GlyfsTer18) in KY. The variant segregated with the phenotype and was absent from public databases and 100 ethnically matched controls. We confirm a novel homozygous KY variant causing a complex neurological phenotype in this family. A review of previously reported KY variants suggests that variants in this gene can cause a spectrum of neurological phenotypes.

Genetic causes of moderate to severe hearing loss point to modifiers.

The genetic underpinnings of recessively inherited moderate to severe sensorineural hearing loss are not well understood, despite its higher prevalence in comparison to profound deafness. We recruited 92 consanguineous families segregating stable or progressive, recessively inherited moderate or severe hearing loss. We utilized homozygosity mapping, Sanger sequencing, targeted capture of known deafness genes with massively parallel sequencing and whole exome sequencing to identify the molecular basis of hearing loss in these families. Variants of the known deafness genes were found in 69% of the participating families with the SLC26A4, GJB2, MYO15A, TMC1, TMPRSS3, OTOF, MYO7A and CLDN14 genes together accounting for hearing loss in 54% of the families. We identified 20 reported and 21 novel variants in 21 known deafness genes; 16 of the 20 reported variants, previously associated with stable, profound deafness were associated with moderate to severe or progressive hearing loss in our families. These data point to a prominent role for genetic background, environmental factors or both as modifiers of human hearing loss severity.

A novel OPA3 mutation revealed by exome sequencing: an example of reverse phenotyping.

IMPORTANCE: We sought to unravel the genetic cause in a consanguineous Pakistani family with a complex neurological phenotype. OBSERVATIONS: Neurological and ophthalmological examination, including videotaping and fundoscopy, and genetic investigations, including homozygosity mapping and exome sequencing, were performed at the University of the Punjab and the University of Lübeck. Participants included 2 severely affected cousins from consanguineous parents, 10 of their reportedly unaffected relatives, and 342 Pakistani controls. Motor symptoms in the 2 patients started at the age of 3 to 4 years and included chorea, cerebellar ataxia, dystonia, and pyramidal tract signs. Genome-wide genotyping delineated 2 regions of homozygosity on chromosomes 13q12.11 to 13q12.13 and 19q12 to 19q13.41. Exome sequencing revealed 2 rare, homozygous variants (c.32 T>A [p.L11Q] in OPA3 and c.941 C>G [p.A314G] in TSHZ3) that segregated with the disease. Only the OPA3 variant was absent in the control subjects and predicted to be damaging. Subsequent ophthalmological assessment revealed bilateral optic atrophy in both patients. CONCLUSIONS AND RELEVANCE: Mutations in OPA3 have been reported in Costeff optic atrophy syndrome. We identify a novel missense mutation in OPA3 as the cause of a complex neurological disorder, expanding the OPA3 -linked phenotype by early-onset pyramidal tract signs and marked lower limb dystonia. Investigation of optic atrophy was initiated only after genetic analysis, a phenomenon referred to as reverse phenotyping.

The c.42_52del11 mutation in TPRN and progressive hearing loss in a family from Pakistan.

The DFNB79 locus harbors TPRN mutations in which have been reported in a few families with deafness. Four frameshift mutations in TPRN have been described to cause severe or severe-to-profound hearing loss in Moroccan and Pakistani families, and a single frameshift mutation was associated with progressive hearing loss in deaf individuals in a Dutch family. We identified a Pakistani family in which the affected individuals were homozygous for a pathogenic mutation, c.42_52del11, in TPRN (p.G15Afs150X). In contrast to the previously reported individuals affected by the same mutation, hearing loss is likely to be progressive in this family. Thus the same mutation of TPRN can be associated with different thresholds of hearing as well as differences in the stability of the phenotype.

Amplification of GC-rich DNA for high-throughput family-based genetic studies.

Researchers face a significant problem in PCR amplification of DNA fragments with high GC contents. Analysis of these regions is of importance since many regulatory regions of different genes and their first exons are GC-rich. There are a large number of protocols for amplification of GC-rich DNA, some of which perform well but are costly. Most of the economical protocols fail to perform consistently, especially on products with >80 % GC contents and a size of >300 bp. One of these protocols requires multiple additions of DNA polymerase during thermal cycling which therefore rules out its utility if a large number of samples have to be amplified. We have established a method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents. These amplicons have GC contents ranging from 65-85 % and sizes up to 870 bp. The protocol uses a PCR buffer containing co-solvents including 2-mercaptoethanol and bovine serum albumin for amplification of DNA. A specific thermal cycling profile is also used which incorporates a high annealing temperature in the first 7 cycles of the reactions. The PCR products are suitable for different molecular biology applications including sequencing.

A p.C343S missense mutation in PJVK causes progressive hearing loss.

Mutations in PJVK, encoding Pejvakin, cause autosomal recessive nonsyndromic hearing loss in humans at the DFNB59 locus on chromosome 2q31.2. Pejvakin is involved in generating auditory and neural signals in the inner ear. We have identified a consanguineous Pakistani family segregating sensorineural progressive hearing loss as a recessive trait, consistent with linkage to DFNB59. We sequenced PJVK and identified a novel missense mutation, c.1028G>C in exon 7 (p.C343S) co-segregating with the phenotype in the family. The p.C343 residue is fully conserved among orthologs from different vertebrate species. We have also determined that mutations in PJVK are not a common cause of hearing loss in families with moderate to severe hearing loss in Pakistan. This is the first report of PJVK mutation in a Pakistani family and pinpoints an important residue for PJVK function.

Prioritized sequencing of the second exon of MYO15A reveals a new mutation segregating in a Pakistani family with moderate to severe hearing loss.

Mutations in MYO15A are associated with deafness in humans, and shaker 2 mice also exhibit a hearing loss due to defects of unconventional myosin 15a. We ascertained a consanguineous Pakistani family with recessively inherited moderate to severe hearing loss, which putatively segregated with markers linked to the DFNB3 locus. Prioritized sequencing of the second exon of MYO15A from the DNA of all affected individuals of family revealed a duplication of Cytosine in a stretch of seven repetitive C nucleotides (c.1185dupC). This mutation results in a frameshift and incorporates a stop codon in the open reading frame of MYO15A (p.E396fsX431). The findings of less severe hearing loss in families with linkage to DFNB3 are only reported for some individuals with mutations in exon 2 of MYO15A, which are further supported by this study. Therefore, on basis of linkage data and the presence of a less severe hearing loss phenotype, sequencing of a single exon of MYO15A can efficiently identify the causative mutations in patients from these families.

An unusual neurological syndrome of crawling gait, dystonia, pyramidal signs, and limited speech.

BACKGROUND: The purpose of the study was to identify and molecularly characterize a neurological syndrome in a consanguineous Pakistani family. METHODS: Five patients, their 2 siblings, and their parents were clinically examined. DNA from all 7 siblings was genotyped with Affymetrix SNP arrays and sequencing of selected candidate genes. RESULTS: An unusual neurological syndrome of crawling gait, predominant leg dystonia, pyramidal signs, microcephaly, and suspected deafness segregated in the family. Three patients ambulated on hands and knees, either by hopping and crossing their legs, or by dragging the legs behind them. Two patients have acquired the ability to walk bipedally with a dystonic gait. Unexpectedly, no chromosomal region was homozygous in patients only. Under different disease models, we localized 7 chromosomal regions in the genome common to all patients. No pathogenic mutations were identified in selected candidate genes or the mitochondrial genome. CONCLUSION: We describe an unusual movement disorder syndrome reminiscent of but distinct from Uner Tan syndrome.

Mutations in CLDN14 are associated with different hearing thresholds.

Mutations in CLDN14, encoding tight junction protein claudin 14, cause profound deafness in mice and humans. We identified a Pakistani family, in which the affected individuals were homozygous for a known pathogenic mutation c.254 T>A resulting in p.V85D substitution in CLDN14; however, in contrast to the previously reported families with mutations in CLDN14, most of the affected individuals in this family exhibit only a severe hearing loss (HL). In order to identify the contribution of CLDN14 to less than profound deafness, we screened for mutations of CLDN14 in 30 multiplex and 57 sporadic cases with moderately severe to severe HL from Pakistan. We identified one other affected individual homozygous for p.V85D substitution. Comparison of audiometric data from all patients indicates that mutations in CLND14 cause varying degrees of HL, which may be enhanced at high frequencies. This suggests that a modifier can reduce the severity of HL associated with mutations of CLDN14. Our data indicate that mutations in CLDN14 should be explored when considering the etiology of less severe HL.