Coursework: the exome of a child with “Rienhoff syndrome”

Bea Rienhoff was born with features that overlap Marfan and Loeys–Dietz syndromes but fitted neither. Her father, a physician and biologist, spent years searching for the cause, and in 2013 her exome sequence identified it. You will repeat the analysis: call variants in her exome yourself, filter and annotate them to find the likely cause, check the evidence in the reads, and show the affected position in the protein structure.

Laboratory report · 2,000 wordsRead the coursework brief on your course page

Patient data – please read

The BAM file is a real patient's sequence. Do not share it outside your studies and do not upload it to public servers. This workbench reads the file on your own computer: samtools, bcftools and IGV all run inside this web page, and the file is never uploaded. Only two things leave your computer, and only when you ask: pressing Annotate with VEP sends the positions and alleles of your filtered variants (no reads, no names) to Ensembl – the same as pasting a VCF into the VEP web page – and the database links in a variant's details open those websites with that variant's position or identifier.

Before you start

Read the news article (ReinhoffSyndromeNewsArticle.pdf, Maher 2013 Nature 498:418–419) and the coursework brief.
Download the coursework data from your course page and unzip it. You need daughters-reads.bam and daughters-reads.bai (keep them together). The reads cover chromosomes 14 and 18, aligned to hg19.
Work through Reads to variants and Variants to meaning first: this workbench uses the same commands and tools.

The workflow

A · load & check→B · call variants→C · annotate→D · filter & prioritise→E · inspect in IGV→F · interpret→G · structure

Step	You will…	For your report
	Load the BAM file; summarise the reads and coverage	Methods; data quality
	Run bcftools; filter the calls	Methods; numbers for the filtering table
	Annotate your calls with Ensembl VEP	Methods; supplementary table
	Filter to a short list of candidate variants and justify each step	Results: filtering table
	Check each candidate in the reads; make IGV figures	Results: variant table and figures
	Research the candidates in the databases and literature	Results and discussion
	Map the protein change onto a 3D structure	Results: structure figure
	Checklist for the report	Everything

Keep a lab notebook

The notes boxes in each step are saved in this browser. Use them to record numbers, commands and observations as you go, and press My notes (top right) to download them. Files you make exist only in this browser tab: use download FILE in the terminal to keep copies. Your Methods section must say exactly what you did – tools, versions, options and thresholds.

Your name or student number (goes on your downloaded notes):

F · Interpret the candidates

For each candidate, gather the evidence – the details card in the Variants tab links to most of these:

Question	Where to look
Is it known? Has it been reported in patients?	dbSNP (rs number), ClinVar, the literature (PubMed)
How common is it in healthy people? How often does the gene tolerate damaging variants?	gnomAD (the website's v2.1 dataset uses GRCh37 positions; v4 uses GRCh38): allele frequency; the gene page's constraint scores (e.g. pLI/LOEUF for loss-of-function, missense Z)
What does the gene do? Which disorders is it linked to, and how are they inherited?	OMIM, UniProt (Function, Disease & Variants), GeneCards
What does the change do to the protein?	VEP consequence and HGVS; SIFT, PolyPhen-2, CADD, SpliceAI; the protein's domains and structure
For a stop-gained variant: will the mRNA be destroyed (nonsense-mediated decay) or a short protein made? Are other stop-gained variants in this gene common in healthy people?	Where the stop falls (which exon, and how far from the last exon); the loss-of-function variants listed for the gene in gnomAD, and how common they are

Notes: the evidence for and against each candidate, with database identifiers (rs numbers, OMIM numbers) for your references.

G · Show the variant in the protein structure

Step G1 · Find and load a structure

Find experimental structures of your protein: its UniProt entry, section Structure (or the PDB). Choose one that contains the region with your variant. (The coursework data folder also contains one structure file – which protein is it?)
Type the four-character PDB code into the PDB ID box of the 3D viewer and press Load (or use Open file for a .pdb file from your computer).
Identify the chains: which chain is your protein? Is there more than one copy, or a partner protein? Hover over the structure and use the workspace list.

Numbering (Box 2 of the brief)

Positions from IGV and VEP count from the first amino acid of the precursor protein made from the gene. Many secreted proteins are cut after translation, and structures of the mature protein are often numbered from its own first residue. Check UniProt's PTM/Processing section: if the mature chain starts at residue n, a precursor position p becomes p − (n − 1) in the structure. Always check that the residue you highlight is the expected amino acid.

Step G2 · Make the figure

Select your residue with the viewer's command line, e.g. select A:123, then show sticks sele, color orange sele, label sele and center sele (use your own chain and number).
Show what it touches: select near sele 4.5 then show sticks sele. Which atoms are within bonding distance – hydrogen bonds, salt bridges, disulfide bonds, contacts with another chain or a ligand? Hover over atoms to identify them.
Add a key and labels, then save a high-resolution image (Save image tab). Write a legend that gives the PDB code, the chains shown and the numbering you used.

Notes: PDB code and resolution; chain(s); the residue number in the gene and in the structure; where the residue is (surface or core, structured or flexible, what it contacts); how the new amino acid differs (size, charge, bonds it can or cannot make); what that may do to the protein.

H · Writing the report

Section	Checklist
Introduction	Marfan and Loeys–Dietz syndromes; the TGF-β pathway; why exome sequencing; the aim
Materials and methods	The data (exome, reads, hg19, how aligned); every tool with its version and the key options/thresholds: samtools 1.17 and bcftools 1.10 (run in the browser workbench), Ensembl VEP (GRCh37; Ensembl release and date), gnomAD v4.1 (via VEP), IGV, the structure viewer and PDB entry; databases consulted
Results	Data quality (coverage); a filtering table (steps and numbers); a variant table (chromosome, position, reference/alternative, zygosity and read counts, gene, HGVS c. and p., consequence, frequency, predictions, identifiers); IGV figures of each variant; the structure figure; the evidence for each candidate
Conclusion	Your conclusion and its strength; the limitations (only two chromosomes, capture and coverage, quality scores, no parental data, predictions are not proof); what further work or tools would confirm it; is the name “Rienhoff syndrome” justified?
References	At most 10 papers, plus database identifiers (OMIM, rs numbers, PDB codes)

Every figure needs a legend that says what is shown, where (genome build and coordinates, or PDB code and residues) and how it was made.

Credits

samtools 1.17 and bcftools 1.10 (Danecek P et al. (2021) GigaScience 10:giab008) compiled to WebAssembly by biowasm, run with Aioli. Genome browser: igv.js (Robinson JT et al. (2023) Bioinformatics 39:btac830). Annotation: Ensembl VEP (McLaren W et al. (2016) Genome Biol 17:122). Molecular graphics: NGL Viewer (Rose AS et al. (2018) Bioinformatics 34:3755–3758). All MIT or Apache licensed.
Reference sequence: UCSC hg19 (GRCh37) chromosomes 14 and 18; gene models: NCBI RefSeq via UCSC.
Case: Maher B (2013) Nature 498:418–419; Rienhoff HY Jr et al. (2013) Am J Med Genet A 161A:2040–2046.

Your data

Load the two coursework files. They are read here, on your computer – nothing is uploaded.

Also available

File	What it is
`ref/hg19.fa`	The hg19 reference for chromosomes 14 and 18 (sequence kept near the exome targets; N elsewhere), with its index
`results/`	Where your output files go (in the Terminal)

If a file will not load: check that you unzipped the download, that you chose both the .bam and the .bai, and that you are using Chrome, Edge, Firefox or Safari on a laptop or desktop computer.