The pathogenic mechanism of syndactyly type V identified in a Hoxd13Q50R knock-in mice.

Syndactyly type V (SDTY5) is an autosomal dominant extremity malformation characterized by fusion of the fourth and fifth metacarpals. In the previous publication, we first identified a heterozygous missense mutation Q50R in homeobox domain (HD) of HOXD13 in a large Chinese family with SDTY5. In order to substantiate the pathogenicity of the variant and elucidate the underlying pathogenic mechanism causing limb malformation, transcription-activator-like effector nucleases (TALEN) was employed to generate a Hoxd13Q50R mutant mouse. The mutant mice exhibited obvious limb malformations including slight brachydactyly and partial syndactyly between digits 2-4 in the heterozygotes, and severe syndactyly, brachydactyly and polydactyly in homozygotes. Focusing on BMP2 and SHH/GREM1/AER-FGF epithelial mesenchymal (e-m) feedback, a crucial signal pathway for limb development, we found the ectopically expressed Shh, Grem1 and Fgf8 and down-regulated Bmp2 in the embryonic limb bud at E10.5 to E12.5. A transcriptome sequencing analysis was conducted on limb buds (LBs) at E11.5, revealing 31 genes that exhibited notable disparities in mRNA level between the Hoxd13Q50R homozygotes and the wild-type. These genes are known to be involved in various processes such as limb development, cell proliferation, migration, and apoptosis. Our findings indicate that the ectopic expression of Shh and Fgf8, in conjunction with the down-regulation of Bmp2, results in a failure of patterning along both the anterior-posterior and proximal-distal axes, as well as a decrease in interdigital programmed cell death (PCD). This cascade ultimately leads to the development of syndactyly and brachydactyly in heterozygous mice, and severe limb malformations in homozygous mice. These findings suggest that abnormal expression of SHH, FGF8, and BMP2 induced by HOXD13Q50R may be responsible for the manifestation of human SDTY5.

Panoramic variation analysis of a family with neurodevelopmental disorders caused by biallelic loss-of-function variants in TMEM141, DDHD2, and LHFPL5.

Highly clinical and genetic heterogeneity of neurodevelopmental disorders presents a major challenge in clinical genetics and medicine. Panoramic variation analysis is imperative to analyze the disease phenotypes resulting from multilocus genomic variation. Here, a Pakistani family with parental consanguinity was presented, characterized with severe intellectual disability (ID), spastic paraplegia, and deafness. Homozygosity mapping, integrated single nucleotide polymorphism (SNP) array, whole-exome sequencing, and whole-genome sequencing were performed, and homozygous variants in TMEM141 (c.270G>A, p.Trp90*), DDHD2 (c.411+767_c.1249-327del), and LHFPL5 (c.250delC, p.Leu84*) were identified. A Tmem141p.Trp90*/p.Trp90* mouse model was generated. Behavioral studies showed impairments in learning ability and motor coordination. Brain slice electrophysiology and Golgi staining demonstrated deficient synaptic plasticity in hippocampal neurons and abnormal dendritic branching in cerebellar Purkinje cells. Transmission electron microscopy showed abnormal mitochondrial morphology. Furthermore, studies on a human in vitro neuronal model (SH-SY5Y cells) with stable shRNA-mediated knockdown of TMEM141 showed deleterious effect on bioenergetic function, possibly explaining the pathogenesis of replicated phenotypes in the cross-species mouse model. Conclusively, panoramic variation analysis revealed that multilocus genomic variations of TMEM141, DDHD2, and LHFPL5 together caused variable phenotypes in patient. Notably, the biallelic loss-of-function variants of TMEM141 were responsible for syndromic ID.

Phenotypic Characterization of Intellectual Disability Caused by MBOAT7 Mutation in Two Consanguineous Pakistani Families.

A homozygous in-frame deletion (c. 758_778del; p. Glu253_Ala259del) in membrane-bound O-acyltransferase family member 7 (MBOAT7), also known as lysophosphatidylinositol acyltransferase (LPIAT1), was previously reported to be the genetic cause of intellectual disability (ID) in consanguineous families from Pakistan. Here, we identified two additional Pakistani consanguineous families with severe ID individuals sharing the same homozygous variant. Thus, we provide further evidence to support this MBOAT7 mutation as a potential founder variant. To understand the genotype-phenotype relationships of the in-frame deletion in the MBOAT7 gene, we located the variant in the fifth transmembrane domain of the protein and determined that it causes steric hindrance to the formation of an α-helix and hydrogen bond, possibly influencing its effectiveness as a functional transmembrane protein. Moreover, extensive neuropsychological observations, clinical interviews and genetic analysis were performed on 6 patients from the 2 families. We characterized the phenotype of the patients and noted the serious outcome of severe paraplegia. Thus, optimal management for symptom alleviation and appropriate screening in these patients are crucial.

Correction to: Homozygous missense variant in the TTN gene causing autosomal recessive limb-girdle muscular dystrophy type 10.

Please be advised that following publication of the original article [1], the authors have identified the following errors with the scientific content.

Novel Compound Heterozygous Mutations in TTI2 Cause Syndromic Intellectual Disability in a Chinese Family.

Telomere maintenance 2 (TELO2)-interacting protein 2 (TTI2) interacts with TTI1 and TELO2 to form the Triple T complex, which is required for various cellular processes, including the double-strand DNA break response, nonsense-mediated mRNA decay, and telomerase assembly. Herein, we identified compound heterozygous mutations in TTI2 using whole-exome sequencing (WES) in a Chinese family with a recessive inheritance pattern of syndromic intellectual disability. The patients displayed intellectual disability, aggressive and self-injurious behaviors, facial dysmorphic features, microcephaly, and skeletal anomalies. In addition, one patient showed cerebral white matter abnormality. Maternal novel indel mutation resulted in a premature termination codon and nonsense-mediated mRNA decay. Paternal reported c.1100C > T mutation changed the highly conserved proline to leucine that located in the DUF2454 domain. Immunoblotting experiments showed significantly decreased TTI2, TTI1, and TELO2 in the patients' lymphocytes. These results indicated that TTI2 loss-of-function mutations might cause an autosomal-recessive syndromic intellectual disability by affecting the Triple T complex. Our report expands the genetic causes of syndromic intellectual disability in the Chinese population.

Homozygous missense variant in the TTN gene causing autosomal recessive limb-girdle muscular dystrophy type 10.

BACKGROUND: Limb-girdle muscular dystrophies (LGMDs) are large group of heterogeneous genetic diseases, having a hallmark feature of muscle weakness. Pathogenic mutations in the gene encoding the giant skeletal muscle protein titin (TTN) are associated with several muscle disorders, including cardiomyopathy, recessive congenital myopathies and limb-girdle muscular dystrophy (LGMD) type10. The phenotypic spectrum of titinopathies is expanding, as next generation sequencing (NGS) technology makes screening of this large gene possible. AIM: This study aimed to identify the pathogenic variant in a consanguineous Pakistani family with autosomal recessive LGMD type 10. METHODS: DNA from peripheral blood samples were obtained, whole exome sequencing (WES) was performed and several molecular and bioinformatics analysis were conducted to identify the pathogenic variant. TTN coding and near coding regions were further amplified using PCR and sequenced via Sanger sequencing. RESULTS: Whole exome sequencing analysis revealed a novel homozygous missense variant (c.98807G > A; p.Arg32936His) in the TTN gene in the index patients. No heterozygous individuals in the family presented LGMD features. The variant p.Arg32936His leads to a substitution of the arginine amino acid at position 32,936 into histidine possibly causing LGMD type 10. CONCLUSION: We identified a homozygous missense variant in TTN, which likely explains LGMD type 10 in this family in line with similar previously reported data. Our study concludes that WES is a successful molecular diagnostic tool to identify pathogenic variants in large genes such as TTN in highly inbred population.

Sequence variants in genes causing nonsyndromic hearing loss in a Pakistani cohort.

BACKGROUND: Hearing loss or hearing impairment is a clinically and genetically heterogeneous disorder. More than 117 genes were discovered to date in hereditary, nonsyndromic hearing loss (NSHL). Identifying novel gene variants and their frequency in specific populations is valuable for public health and potentially for genetic screening of NSHL. AIMS: To identify the gene variants underlying NSHL in a Pakistani cohort. METHODS AND RESULTS: A cohort of 40 school-aged children with NSHL was initially screened for variants in GJB2, the gene with the highest incidence of variants in other populations with NSHL. We found known homozygous as well as compound heterozygous GJB variants in 15 individuals. Next, we used targeted next generation sequencing (TNGS) for the remaining 25 individuals and identified 20 different variants in 14 genes (SLC26A4, KCNQ4, MYO7A, MYO15A, TMPRSS3, ESPN, TMC1, GIPC3, LHFPL5, WFS1, DFNB59, GRXCR1, ESRRB, and LRTOMT). CONCLUSIONS: We described common and novel variants in 15 genes in a Pakistani cohort of NSHL.

A Novel Homozygous Nonsense Mutation p.Cys366* in the WNT10B Gene Underlying Split-Hand/Split Foot Malformation in a Consanguineous Pakistani Family.

Split hand/split foot malformation (SHFM) or ectrodactyly is characterized by a deep median cleft of the hand or foot, hypoplasia or aplasia of the metacarpals, metatarsals, and phalanges. It is a clinically and genetically heterogeneous group of limb malformations. This study aimed to identify the pathogenic variant in a consanguineous Pakistani family with autosomal recessive SHFM. Peripheral blood samples were obtained, DNA was extracted, WNT10B coding and noncoding regions were PCR amplified and Sanger sequencing was performed using workflow suggested by Thermo Fisher Scientific. A novel homozygous nonsense variant (c.1098C>A; p.Cys366*) was identified in the WNT10B gene in the index patients, which probably explains SHFM type 6 in this family in comparison with similar data from the literature.

Rare compound heterozygous mutations in gene MSH6 cause constitutive mismatch repair deficiency syndrome.

Few studies reported patients who harbored three kinds of primary tumors simultaneously. Here, we present a 9-year-old boy with colon carcinoma, brain medulloblastoma, and lymphoma. Genetic mutation detection was explored with next-generation sequencing, and compound heterozygous mutations in gene MSH6 c.3103C>T p.Arg1035Ter and c.3261dupC p.Phe1088LeufsTer were discovered.

Clinical and genetic characteristics of Chinese patients with familial or sporadic pediatric cataract.

BACKGROUND: Pediatric cataract is a clinically and genetically heterogeneous disease which is a significant cause of lifelong visual impairment and treatable blindness. Our study aims to investigate the genotype spectrum in a group of Chinese patients with pediatric cataract. METHODS: We enrolled 39 families with pediatric cataract from October 2015 to April 2016. DNA samples of the probands were analyzed by target next-generation sequencing. Variants were validated using Sanger sequencing in the probands and available family members. RESULTS: In our cohort of 39 cases with different types of pediatric cataract, 23 cases were found to harbor putative pathogenic variants in 15 genes: CRYAA, CRYBA1, CRYBA4, CRYBB1, CRYGC, CRYGD, MIP, GCNT2, IARS2, NHS, BCOR, BFSP2, FYCO1, MAF, and PAX6. The mutation detection rates in the familial and sporadic cases were 75 and 47.8%, respectively. Of the 23 causative variants, over half were novel. CONCLUSIONS: This is a rare report of systematic mutation screening analysis of pediatric cataract in a comparably large cohort of Chinese patients. Our observations enrich the mutation spectrum of pediatric cataract. Next-generation sequencing provides significant diagnostic information for pediatric cataract cases, especially when considering sporadic and subtle syndromal cases.

Identification of a Novel Nonsense ASPM Mutation in a Large Consanguineous Pakistani Family Using Targeted Next-Generation Sequencing.

AIMS: To identify the pathogenic mutation underlying microcephaly primary hereditary (MCPH) in a large consanguineous Pakistani family. METHODS: A five-generation family with an autosomal recessive transmission of MCPH was recruited. Targeted next-generation DNA sequencing was carried out to analyze the genomic DNA sample from the proband with MCPH using a previously designed panel targeting 46 known microcephaly-causing genes. Sanger sequencing was performed to verify all identified variants. RESULTS: We found a novel homozygous nonsense mutation, c.7543C>T, in the ASPM gene. This mutation led to the substitution of an arginine with a stop codon at amino acid residue 2515 (p.Arg2515Ter). The mutation cosegregated with the MCPH phenotype in all affected and obligate carrier family members, but was not present in public databases (dbSNP147, Exome Variant Server, the 1000 Genomes Project, Exome Aggregation Consortium, Human Gene Mutation Database, and ClinVar) or 200 control individuals. The c.7543C>T mutation in ASPM may activate nonsense-mediated mRNA decay pathways and could underlie the pathogenesis of MCPH through a loss-of-function mechanism. CONCLUSIONS: The c.7543C>T (p.Arg2515Ter) mutation in ASPM is a novel pathogenic mutation for the typical MCPH phenotype in this family.

A novel homozygous missense mutation in BHLHA9 causes mesoaxial synostotic syndactyly with phalangeal reduction in a Pakistani family.

Mesoaxial synostotic syndactyly with phalangeal reduction (MSSD) is a rare non-syndromic limb malformation with autosomal recessive inheritance. To date, only a few affected families with MSSD who had BHLHA9 mutations have been reported. The present report describes a consanguineous Pakistani family with five affected individuals with MSSD who exhibited an autosomal recessive pattern. Genotyping followed by Sanger sequencing was performed, and we identified a novel homozygous missense mutation (c.311T>C, p.Ile104Thr) in the BHLHA9 gene. This finding expands the spectrum of known mutations in the BHLHA9 gene that cause MSSD.

Molecular Analysis of Twelve Pakistani Families with Nonsyndromic or Syndromic Hearing Loss.

AIM: To investigate the causative genetic mutations in 12 Pakistani families with nonsyndromic or syndromic hearing loss. METHODS: Mutations in the most common causative gene for hearing loss, GJB2, were evaluated by Sanger sequencing. Targeted next-generation sequencing or whole-exome sequencing was used to analyze the genomic DNA samples from 11 probands with hearing loss. Sanger sequencing was performed to verify all identified variants. RESULTS: We found pathogenic, or likely to be pathogenic, mutations in all 12 families, including six known mutations in GJB2, SLC26A4, LHFPL5, and USH2A and eight novel mutations in ESPN, MYO7A, LRTOMT, PCDH15, USH2A, or EPS8L2. Notably, four compound heterozygous mutations in the MYO7A and USH2A genes were detected in two consanguineous families. In addition, the novel frameshift mutation in EPS8L2 was first documented in Pakistan. CONCLUSIONS: Our study increases the spectrum of mutations associated with hearing loss in the Pakistani population. In addition, our study highlights the fact that compound heterozygous mutations, although rare, can occur in consanguineous families.

Molecular analysis of 23 Pakistani families with autosomal recessive primary microcephaly using targeted next-generation sequencing.

Primary microcephaly is genetically heterogeneous, with most cases showing autosomal recessive inheritance. We designed a panel containing 46 primary microcephaly-causing genes and performed mutation screening in 23 Pakistani families with autosomal recessive primary microcephaly. We found mutations that were pathogenic or likely to be pathogenic in 22 families, including 18 families with known mutations in ASPM, three with novel mutations in WDR62 and one with a novel in-frame deletion mutation in CASC5. Affected individuals harbored the c.3978G>A (p.W1326*) ASPM mutation in 15 families (nine consanguineous and six non-consanguineous), suggesting a high carrier rate of the nonsense mutation in Pakistani individuals. We identified three novel homozygous WDR62 mutations, including an intragenic deletion of 10 299 bp, a splicing mutation and a nonsense mutation. Our results confirm that mutations in ASPM or WDR62 are the major cause of autosomal recessive primary microcephaly in the Pakistani population.

Intermittent cold exposure results in visceral adipose tissue "browning" in the plateau pika (Ochotona curzoniae).

The plateau pika has developed tolerance to cold and hypoxia in order to adapt to living in the extreme environment of the Qinghai-Tibetan Plateau. One mammalian mechanism for cold adaptation is thermogenesis by brown adipose tissue (BAT), but the degree to which pika exploits this mechanism or how it may be modified by the additional stresses of high altitude is not known. Intermittent Cold Exposure (ICE) is an approachable method to study cold adaptation in rodents. To investigate the role of adipose tissue in the adaptation of pika to cold temperatures, we have studied pika during ICE. We find that pika kept in warm temperatures has little classical brown fat, but "browning" of white adipose tissues is observed rapidly upon cold exposure. This is demonstrated by the increased expression of several markers of brown fat differentiation including uncoupling protein 1 (UCP-1). Surprisingly, this occurs mainly in visceral rather than epididymal adipose tissue. In addition, ICE increases the expression of several general adipose differentiation markers at both the mRNA and protein levels. These substantial changes in the distribution of fat are accomplished without changes in weight or blood levels of glucose and triglycerides, suggesting that the adaptable changes are coordinated and self-compensated. Together, our results demonstrate that ICE promotes recruitment of BAT in pika, and unlike small mammals in at lower altitudes, pika can activate visceral WAT to adapt to cold stress without major changes overall energy balance.