Identification of a pathogenic variant and pre-implantation genetic testing for a Chinese family affected with split-hand/foot malformation.

Split-hand/foot malformation is a serious congenital limb malformation characterized by syndactyly and underdevelopment of the phalanges and metatarsals. In this study, we reported a case of a fetus with hand-foot cleft deformity. Whole exome and Sanger sequencing were used to filter out candidate gene mutation sites and provide pre-implantation genetic testing(PGT) for family members. Genetic testing results showed that there was a homozygous mutation c.786G>A (p.Trp262*) in the fetal WNT10B, and both parents were carriers of heterozygous mutations. PGT results showed that out of the two blastocysts, one was a heterozygous mutant and the other was a homozygous mutant. All the embryos had diploid chromosomes. The heterozygous embryo was transferred, and a singleton pregnancy was successfully achieved. This study suggests that homozygous mutations in WNT10B are the likely cause of hand-foot clefts in this family. For families with monogenic diseases, preimplantation genetic testing can effectively prevent the birth of an affected child only after identifying the pathogenic mutation.

Human split hand/foot variants are not as functional as wildtype human PRDM1 in the rescue of craniofacial defects.

BACKGROUND: Split hand/foot malformation (SHFM) is a congenital limb disorder presenting with limb anomalies, such as missing, hypoplastic, or fused digits, and often craniofacial defects, including a cleft lip/palate, microdontia, micrognathia, or maxillary hypoplasia. We previously identified three novel variants in the transcription factor, PRDM1, that are associated with SHFM phenotypes. One individual also presented with a high arch palate. Studies in vertebrates indicate that PRDM1 is important for development of the skull; however, prior to our study, human variants in PRDM1 had not been associated with craniofacial anomalies. METHODS: Using transient mRNA overexpression assays in prdm1a-/- mutant zebrafish, we tested whether the PRDM1 SHFM variants were functional and could lead to a rescue of the craniofacial defects observed in prdm1a-/- mutants. We also mined previously published CUT&RUN and RNA-seq datasets that sorted EGFP-positive cells from a Tg(Mmu:Prx1-EGFP) transgenic line that labels the pectoral fin, pharyngeal arches, and dorsal part of the head to examine Prdm1a binding and the effect of Prdm1a loss on craniofacial genes. RESULTS: The prdm1a-/- mutants exhibit craniofacial defects including a hypoplastic neurocranium, a loss of posterior ceratobranchial arches, a shorter palatoquadrate, and an inverted ceratohyal. Injection of wildtype (WT) hPRDM1 in prdm1a-/- mutants partially rescues the palatoquadrate phenotype. However, injection of each of the three SHFM variants fails to rescue this skeletal defect. Loss of prdm1a leads to a decreased expression of important craniofacial genes by RNA-seq, including emilin3a, confirmed by hybridization chain reaction expression. Other genes including dlx5a/dlx6a, hand2, sox9b, col2a1a, and hoxb genes are also reduced. Validation by real-time quantitative PCR in the anterior half of zebrafish embryos failed to confirm the expression changes suggesting that the differences are enriched in prx1 expressing cells. CONCLUSION: These data suggest that the three SHFM variants are likely not functional and may be associated with the craniofacial defects observed in the humans. Finally, they demonstrate how Prdm1a can directly bind and regulate genes involved in craniofacial development.

Gollop-Wolfgang Complex Is Associated with a Monoallelic Variation in WNT11.

Gollop-Wolfgang complex (GWC) is a rare congenital limb anomaly characterized by tibial aplasia with femur bifurcation, ipsilateral bifurcation of the thigh bone, and split hand and monodactyly of the feet, resulting in severe and complex limb deformities. The genetic basis of GWC, however, has remained elusive. We studied a three-generation family with four GWC-affected family members. An analysis of whole-genome sequencing results using a custom pipeline identified the WNT11 c.1015G>A missense variant associated with the phenotype. In silico modelling and an in vitro reporter assay further supported the link between the variant and GWC. This finding further contributes to mapping the genetic heterogeneity underlying split hand/foot malformations in general and in GWC specifically.

A genotype-phenotype correlation in split-hand/foot malformation type 1: further refinement of the phenotypic subregions within the 7q21.3 locus.

Background: Split-hand/foot malformation type 1 (SHFM1) refers to the group of rare congenital limb disorders defined by the absence or hypoplasia of the central rays of the autopods with or without accompanying anomalies, such as hearing loss, craniofacial malformation, and ectodermal dysplasia. Consequently, the condition is characterized by clinical variability that hinders diagnostic and counseling procedures. SHFM1 is caused by pathogenic variants affecting the DLX5/6 genes and/or their tissue-specific enhancers at the 7q21.3 locus. Herein, we report on seven patients from five unrelated Polish families affected by variable symptoms of the SHFM1 spectrum, all harboring 7q21.3 or 7q21.2-q21.3 rearrangements, and provide a genotype-phenotype correlation in the studied cohort. Methods: We applied GTG banding, array-based comparative genomic hybridization (aCGH), and whole-genome sequencing (WGS) in order to identify the causative aberrations in all affected patients. Results: The identified pathogenic structural variants included deletions and/or translocations involving the 7q21.3 locus, i.e., t(7;10)(q21.3;q22.2) and t(7;12)(q21.3;q21.2) in all affected individuals. Interestingly, a sporadic carrier of the latter aberration presented the SHFM1 phenotype with additional features overlapping with Baker-Gordon syndrome (BAGOS), which resulted from the translocation breakpoint at chromosome 12 within the SYT1 gene. Conclusion: Clinical variability of the studied cohort reflects the composition of the DLX5/6 regulatory elements that were dislocated from their target genes by chromosomal rearrangements. The correlation of our data with the previously published observations enabled us to update the phenotypic subregions and regulatory units within the SHFM1 locus. In addition, we present the first case of SHFM1 and BAGOS-like phenotype that resulted from translocation breakpoints at chromosomes 7 and 12, both of which were pathogenic, and consequently, we show the first evidence that BAGOS can also result from the regulatory loss-of-function SYT1 mutations. In this paper, we emphasize the utility of sequence-based approaches in molecular diagnostics of disorders caused by regulatory structural variants.

A Rare Case Report of Split Hand and Foot Malformation.

Introduction: Authors report a rare case report about split hand and foot malformation (SHFM) also sometimes referred to as ectrodactyly. Case Report: The patient with hand and foot malformations presented to casualty. A 60-year-old male was brought with alleged history of road traffic accident with tenderness and deformity in left thigh. On further physical examination, a malformation was present in bilateral feet and right hand. Plain radiographs were taken after emergency primary management which revealed a fracture of shaft of femur of the left side and absence of 2nd and 3rd phalanges in bilateral feet and lobster claw like malformation in the right hand. The patient was further investigated and operated with femur interlocking nail and later discharged under stable condition. Screening for other congenital defects was done. Conclusion: Patients with SHFM should undergo screening for other congenital anomalies. Electrocardiogram, 2D ECHO, chest radiograph, and ultrasonography abdomen should be done. Genetic analysis ideally should be done to identify mutations involved. Surgical intervention is only required when patient demands improved function of limb.

A complex structural variant near SOX3 causes X-linked split-hand/foot malformation.

Split-hand/foot malformation (SHFM) is a congenital limb defect most typically presenting with median clefts in hands and/or feet, that can occur in a syndromic context as well as in isolated form. SHFM is caused by failure to maintain normal apical ectodermal ridge function during limb development. Although several genes and contiguous gene syndromes are implicated in the monogenic etiology of isolated SHFM, the disorder remains genetically unexplained for many families and associated genetic loci. We describe a family with isolated X-linked SHFM, for which the causative variant could be detected after a diagnostic journey of 20 years. We combined well-established approaches including microarray-based copy number variant analysis and fluorescence in situ hybridization coupled with optical genome mapping and whole genome sequencing. This strategy identified a complex structural variant (SV) comprising a 165-kb gain of 15q26.3 material ([GRCh37/hg19] chr15:99795320-99960362dup) inserted in inverted position at the site of a 38-kb deletion on Xq27.1 ([GRCh37/hg19] chrX:139481061-139518989del). In silico analysis suggested that the SV disrupts the regulatory framework on the X chromosome and may lead to SOX3 misexpression. We hypothesize that SOX3 dysregulation in the developing limb disturbed the fine balance between morphogens required for maintaining AER function, resulting in SHFM in this family.

New pathogenic variant in DLX5: New clues for a clinical spectrum from split-hand-foot malformation to fibular aplasia, tibial campomelia and oligosyndactyly.

Introduction: FATCO (Fibular Aplasia, Tibial Campomelia and Oligosyndactyly) is a very infrequent skeletal dysplasia classified within the limb hypoplasia-reduction defects group whose genetic cause has not yet been identified. The advent of next-generation sequencing is enabling the diagnosis of diseases with no previously known genetic cause. Methods: We performed a thorough autopsy on a fetus whose pregnancy was legally terminated due to severe malformations detected by ultrasound. A trio exome was run to identify the genetic cause and risk of recurrence. Previous literature of similar cases was systematically searched. Results: Anatomopathological analyses revealed complete fibular aplasia, shortened and campomelic tibia, absent ankle joint, club right foot and a split foot malformation, leading to the diagnosis of FATCO. Exome sequencing showed that the female fetus carried a de novo nonsense variant in DLX5. The literature search permitted the collection of information on 43 patients with FATCO, the majority of whom were males diagnosed postnatally. In most cases, lower limbs were affected exclusively, but in 39.5% of cases the upper limbs were also affected. Conclusion: The pathologies associated with DLX5 variants encompass a wide spectrum of manifestations ranging from abnormalities exclusively in the hands and feet to long bones such as the tibia and fibula.

PRDM1 DNA-binding zinc finger domain is required for normal limb development and is disrupted in split hand/foot malformation.

Split hand/foot malformation (SHFM) is a rare limb abnormality with clefting of the fingers and/or toes. For many individuals, the genetic etiology is unknown. Through whole-exome and targeted sequencing, we detected three novel variants in a gene encoding a transcription factor, PRDM1, that arose de novo in families with SHFM or segregated with the phenotype. PRDM1 is required for limb development; however, its role is not well understood and it is unclear how the PRDM1 variants affect protein function. Using transient and stable overexpression rescue experiments in zebrafish, we show that the variants disrupt the proline/serine-rich and DNA-binding zinc finger domains, resulting in a dominant-negative effect. Through gene expression assays, RNA sequencing, and CUT&RUN in isolated pectoral fin cells, we demonstrate that Prdm1a directly binds to and regulates genes required for fin induction, outgrowth and anterior/posterior patterning, such as fgfr1a, dlx5a, dlx6a and smo. Taken together, these results improve our understanding of the role of PRDM1 in the limb gene regulatory network and identified novel PRDM1 variants that link to SHFM in humans.

BHLHA9 homozygous duplication in a consanguineous family: A challenge for genetic counseling.

Split-hand/foot malformation (SHFM) with long-bone deficiency (SHFLD) is a rare condition characterized by SHFM associated with long-bone malformation usually involving the tibia. It includes three different types; SHFLD1 (MIM % 119,100), SHFLD2 (MIM % 610,685) and SHFLD3 (MIM # 612576). The latter was shown to be the most commonly reported with a duplication in the 17p13.1p13.3 locus that was narrowed down to the BHLHA9 gene. Here, we report a consanguineous Lebanese family with three members presenting with limb abnormalities including tibial hemimelia. One of these patients presented with additional bowing fibula and another with bilateral split hand. CGH array analysis followed by RQ-PCR allowed us to detect the first homozygous duplication on the short arm of chromosome 17p13.3 including the BHLHA9 gene and involved in SHFLD3. Interestingly, one patient with the homozygous duplicated region, carrying thus four BHLHA9 copies presented with long bone deficiency but no SHFM. The incomplete penetrance and the variable expressivity of the disease in this family as well as the presence of the BHLHA9 homozygous duplication rendered genetic counseling extremely challenging and preimplantation genetic diagnosis almost impossible.

Variable clinical presentation of split hand/foot malformation syndrome in a family with microduplication of 10q24.32: a case report.

SHFM (Split Hand/Foot Malformation) is a heterogeneous group of disorders characterized by the presence of clefts in the hands and feet, along with syndactyly of the digits. In this article, we describe a family in which two members exhibit characteristic developmental abnormalities associated with SHFM, presenting with variable clinical features. Using whole-genome sequencing, we identified a microduplication of a chromosomal segment on locus 10q24.32, specifically spanning positions 102934495 to 103496555, encompassing genes BTRC, POLL, FBXW4 and LBX1 in the proband. Genomic duplications, including these genes, were previously described in patients diagnosed with the third type of SHFM. We validated the presence of this structural rearrangement in 7 family members, including the proband and the proband's father. Remarkably, further investigation demonstrated that the detected duplication exhibits a mosaic state in the phenotypically normal paternal grandmother of the proband, thereby providing a plausible explanation for the absence of a pathological phenotype in her.

Microduplication of BTRC detected in a Chinese family with split hand/foot malformation type 3.

Split hand/foot malformation (SHFM) is a clinically heterogeneous genetic disorder, which is mainly characterized by median clefts of the hand/feet due to the absence of the central digital rays. Several subgroups of SHFM have been identified, including SHFM1 to SHFM6. SHFM3 is an autosomal dominant disease, which has been identified to associate with a 500 kb microduplication at 10q24. The duplication involved several genes, including LBX1, BTRC, POLL, FBXW4, and so forth. In the study, using trio clinical exome sequencing, a 120 kb microduplication containing only BTRC were identified in a Chinese family affected with SHFM3. Further confirmation was performed using qRT-PCR assay, which showed that the 120 kb duplication was co-segregated with SHFM phenotypes in the family. It is the smallest duplication which has ever been reported relating to SHFM3. Furthermore, the transcription levels of BTRC mRNA in lymphocyte of the proband was significantly higher than that in the healthy control. The study provided evidence for the limb malformation caused by abnormal BTRC expression, and suggested that next generation sequencing could provide more precise diagnosis to SHFM3 patients.

Identification of a novel heterozygous missense TP63 variant in a Chinese pedigree with split-hand/foot malformation.

BACKGROUND: Tumor protein p63 is an important transcription factor regulating epithelial morphogenesis. Variants associated with the TP63 gene are known to cause multiple disorders. In this study, we determined the genetic cause of split-hand/foot malformation in a Chinese pedigree. METHODS: For this study, we have recruited a Chinese family and collected samples from affected and normal individuals of the family (three affected and two normal). Whole exome sequencing was performed to detect the underlying genetic defect in this family. The potential variant was validated using the Sanger sequencing approach. RESULTS: Using whole-exome and Sanger sequencing, we identified a novel heterozygous pathogenic missense variant in TP63 (NM_003722.5: c.921G > T; p.Met307Ile). This variant resulted in the substitution of methionine with isoleucine. Structural analysis suggested a resulting change in the structure of a key functional domain of the p63 protein. CONCLUSION: This novel missense variant expands the TP63 variant spectrum and provides a basis for genetic counseling and prenatal diagnosis of families with split-hand/foot malformation or other TP63-related diseases.

Newborn Male With Fibular Aplasia, Tibial Campomelia, and Oligosyndactyly Syndrome: A New Case Report Putting the Condition Under Spotlight.

The syndrome of fibular aplasia, tibial campomelia, and oligosyndactyly (FATCO syndrome) is a rare genetic disease that has been increasingly reported over the past 40 years. We report the case of a newborn boy with unilateral skeletal abnormalities that were evident clinically and radiologically. The baby was an infant of a diabetic mother, and the Egyptian parents were consanguineous with a strong family history of genetic diseases and congenital anomalies. Besides describing a new case report of this syndrome, we emphasize the importance of prenatal diagnosis and genetic counseling, especially for families at high risk for genetic diseases in developing countries.

Structural model of human PORCN illuminates disease-associated variants and drug-binding sites.

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Based on homology to mammalian MBOAT proteins, we developed and validated a structural model of human PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model enhances our mechanistic understanding of PORCN substrate recognition and catalysis, as well as the inhibition of its enzymatic activity, and can facilitate the development of improved inhibitors and the understanding of disease-relevant PORCN mutants. This article has an associated First Person interview with the joint first authors of the paper.

Homozygous nonsense mutation of WNT10B gene in a Moroccan family with split-hand foot malformation identified by exome sequencing: a case report.

Split-hand foot malformation (SHFM) is a clinically heterogeneous congenital limb defect affecting predominantly the central rays of hands and/or feet. The clinical expression varies in severity between patients as well between the limbs in the same individual. SHFM might be non-syndromic with limb-confined manifestations or syndromic with extra-limb manifestations. Isolated SHFM is a rare condition with an incidence of about 1 per 18,000 live born infants and accounts for 8-17 % of all limb malformations. To date, many chromosomal loci and genes have been described as associated with isolated SHFM, i.e., SHFM1 to 6. SHFM6 is one of the rarest forms of SHFM, and is caused by mutations in WNT10B gene. Less than ten pathogenic variants have been described. We have investigated a large consanguineous Moroccan family with three affected members showing feet malformations with or without split hand malformation phenotypes. Using an exome sequencing approach, we identified a homozygous nonsense variant p.Arg115* of WNT10B gene retaining thereby the diagnosis of SHFM6. This homozygous nonsense mutation identified by exome sequencing in a large family of split hand foot malformation highlights the importance of exome sequencing in genetically heterogeneous entities.

Whole genome sequencing reveals translocation breakpoints disrupting TP63 gene underlying split hand/foot malformation in a Chinese family.

BACKGROUND: Split hand/foot malformation (SHFM) is a congenital limb developmental disorder, which impairs the fine activities of hand/foot in the affected individuals seriously. SHFM is commonly inherited as an autosomal dominant disease with incomplete penetrance. Chromosomal aberrations such as copy number variations and translocations have been linked to SHFM. This study aimed to identify the genetic cause for three patients with bilateral hand and foot malformation in a Chinese family. METHODS: Karyotyping, single-nucleotide polymorphism (SNP) array, whole exome sequencing, whole genome sequencing, and Sanger sequencing were applied to identify the pathogenic variant. RESULTS: Karyotyping revealed that the three patients had balanced reciprocal translocation, 46, XX, t(3;15) (q29;q22). SNP array identified no pathogenic copy number variation in the proband. Trio-WES (fetus-mother-father) sequencing results revealed no pathogenic variants in the genes related to SHFM. Whole-genome low-coverage mate-pair sequencing (WGL-MPS), breakpoint PCR, and Sanger sequencing identified the breakpoints disrupting TP63 in the patients, but not in healthy family members. CONCLUSION: This study firstly reports that a translocation breakpoint disrupting TP63 contributes to the SHFM in a Chinese family, which expands our knowledge of genetic risk and counseling underlying SHFM. It provides a basis for genetic counseling and prenatal diagnosis (preimplantation genetic diagnosis) for this family.

Split hand/foot malformation associated with 20p12.1 deletion: A case report.

Split hand/foot malformation (SHFM) or ectrodactyly is a rare congenital disorder affecting limb development characterized by clinical and genetic heterogeneity. SHFM is usually inherited as an autosomal dominant trait with incomplete penetrance. Isolated and syndromic forms are described. The extent of associated malformations is highly variable and multiple syndromes with clinical and genetic overlap have been described. We report here a 28 year-old man presenting with SHFM, sparse hair and widespread freckles. Array-CGH identified a 450 kb de novo 20p12.1 microdeletion encompassing three exons (exon 6 to 8) of MACROD2. Although MACROD2 mutations have not been associated with limb malformation until now, it is located next to KIF16B, which is involved in fibroblast growth factor receptor (FGFR) signaling. Additionally, the deletion encompassed a histone modification H3K27ac mark, known as a provider of quantitative readout of promoter and enhancer activity during human limb development. Altogether, these findings suggest that the 20p12.1 CNV is causative of SHFM in the present case through disturbance of regulatory elements functioning.

A classification system for split-hand/ foot malformation (SHFM): A proposal based on 3 pedigrees with WNT10B mutations.

SHFM6 (OMIM 225300) is caused by WNT10B pathogenic variants (12q13.12). It is one of the rarest forms of SHFM; with only seven pathogenic variants described in the world literature. Furthermore, it has not been determined if SHFM6 has specific phenotypic characteristics. In this paper, we present a case series of three unrelated families with SHFM6 caused by three novel WNT10B pathogenic variants. The index patient of the first family was homozygous for the nonsense variant c.676C > T (p.Arg226*) in the WNT10B gene. The index case of the second family had a homozygous splice variant c.338-1G > C in the WNT10B gene. Finally, the index case of the third family carried two different variants in the WNT10B gene: A nonsense variant (p.Arg226*), and a missense variant (p.Gln86Pro). The latter represents the first compound heterozygous pathogenic variant related to SHFM6. We also offer a classification system for the hand/foot defects to illustrate the specific phenotypic characteristics of SHFM6. Based on this classification and a review of all previously reported cases, we demonstrate that SHFM6 caused by WNT10B pathogenic variants have the following characteristics: more severe feet defects (compared to the hand defects), polydactyly, severe flexion digital contractures, and phalangeal dysplasia.